Anti-inflammatory properties of marine lipid compositions

ABSTRACT

Novel marine lipid compositions comprising triglycerides and omega-3 rich phospholipids are described. The compositions are characterized by providing highly bioavailable omega-3, increased tissue incorporation of omega-3 and reduced concentration of pro-inflammatory cytokines.

This application claims the benefit of U.S. Provisional Applications60/798,026, 60/798,027, and 60/798,030, all filed May 5, 2006, and U.S.Provisional Application 60/872,096, filed Dec. 1, 2006, each of which isincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel marine lipid compositionscomprising combinations of omega-3 fatty acid rich functionalphospholipids and omega-3 fatty acid rich triglycerides. In addition,food supplements, functional food, drugs and feed products comprisingsuch compositions are provided along with methods of their use.

BACKGROUND OF THE INVENTION

Marine lipids such as omega-3 rich triglycerides and omega-3 richphospholipids can be isolated from a number of different natural sourcessuch as fish, crustaceans, plankton, seals, whales as well as algaeusing extraction technologies. In addition, they can be preparedindustrially using chemical or bio-catalytical methods such as enzymecatalyzed transesterification of crude soy lecithin with fish oil fattyacids [1].

The anti-inflammatory properties of omega-3 fatty acids are well knownand the use as an anti-inflammatory agent has been described both fortriglycerides and phospholipids [2-3]. Actually, omega-3 fatty acids arefamous for their anti-inflammatory properties, and it has been shownthat omega-3 fatty acids alleviate the symptoms of a series ofautoimmune, atherosclerotic and inflammatory diseases includinginflammatory bowel diseases and rheumatoid arthritis [4-6]. Suppressionof inflammation has been proposed as one of the strategies to slow downthe progress of these diseases. Hence, this invention discloses theeffect on marine lipid compositions on the concentration of markers ofinflammation such as TNF-α and other cytokines such as interleukin-1βand interleukin 6. In addition, since arachidonic acid (AA) is thepredominant precursor of the eicosanoid mediators of inflammatoryresponses (prostaglandins, thromboxanes and leukotrienes), thisinvention discloses the reduction of AA level and the improvement in theEPA/AA ratio in different lipid pools in tissues such as in thephospholipids isolated from adipose tissue, heart, testicles, plasma,brain and liver.

The bioavailability of EPA and DHA from fish oil triglycerides have beenreported to be high in healthy adults. However, for certain conditionsi.e. pathological conditions such as extrahepatic cholestasis and forpre-term infants the absorption can be low. For example it was shownthat the absorption of DHA from egg lecithin in pre-term infants was 90%compared to 80% from triglycerides [7]. Absorption of long chain PUFA(AA and DHA) is less (75% and 62%, respectively) than the absorption ofC18 PUFA (94%) in pre-term infants [8]. The difference between C18 PUFAand long chain PUFA absorption is likely to become less apparent inolder children and adults. Sala-Vila et al [9-10] investigated thebioavailabilities of DHA-PL and DHA-TG in full term infants and found nodifferences based on plasma lipid enrichments. Valenzuela et al. [11]supplemented female rats with different forms of DHA including egg yolkPL and single cell algae TG. They found also no difference in absorptionof DHA from PL and TG based on plasma lipid enrichments. However, thetissue and milk fat levels were higher in PL-DHA compared to the TG-DHAsupplemented rats. These data indicate that although there were nodifferences in the bioavailability, efficacy with respect to tissueenrichment was higher for PL-DHA compared to TG-DHA. Furthermore, therelative absorption of EPA and DHA ethyl esters (4 g/d) compared tooleic acid calculated from peak concentrations was 94 and 100%,respectively. Estimates of relative absorption based on the area underthe concentration curve indicated a relative absorption of 91% for EPAand 93% for DHA [12]. Bioavailability of C18:1, C18:2 and C18:3 in adulthumans are close to 100% (note 94% in preterm infants). Thus thebioavailability of EPA and DHA delivered in different forms is,according to previous, work likely to be over 90%.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a composition comprising atriglyceride and a phospholipids in a ratio ranging from 1:10 to 10:1;said phospholipids having the following structure:

wherein R1 is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is amixture of H, choline, ethanolamine, inositol and serine, saidphospholipid having at least 1% of DHA/EPA, said phospholipids have aconcentration of OH in the range of 25-50%. In further embodiments, theinvention provides a marine lipid composition characterized by providinghigher uptake of omega-3 fatty acids into plasma as compared toadministration of purified triglycerides, phospholipids, or naturalmarine phospholipids. In further embodiments, the invention provides acomposition characterized by efficiently improving the AA/EPA ratio inplasma phospholipids as compared to administration of purifiedtriglycerides, phospholipids, or natural marine phospholipids. In stillother embodiments, the invention is a marine lipid compositioncharacterized by efficiently increasing the concentration of omega-3fatty acids in tissues as compared to administration of purifiedtriglycerides, phospholipids, or natural marine phospholipids. In stillfurther embodiments, the invention the composition is characterized byreducing the concentration of biomarkers of inflammation as compared toadministration of purified triglycerides, phospholipids, or naturalmarine phospholipids. In other embodiments of the invention, the marinelipid composition is formulated into an animal feed, a food product, afood supplement and a drug.

In some embodiments, the present invention provides a compositioncomprising phospholipids having the following structure:

wherein R1 is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is amixture of H, choline, ethanolamine, inositol and serine, saidphospholipid having at least 1% of DHA/EPA at positions R1 and/or R2 andfrom about 20-50% of OH at positions R1 and/or R2. In some embodiments,the composition is acylated in a range from about 55% to about 85%. Insome embodiments, the omega-3 fatty acids are selected from the groupconsisting of EPA, DHA, DPA and α-linolenic acid (ALA). In someembodiments, the composition is substantially free of organic solventsand volatile organic compounds such as short chain fatty acids, shortchain aldehydes and short chain ketones. In some embodiments, thecomposition has at least 5% of a combination of EPA and DHA esterified.In some embodiments, the composition has at least 10% of a combinationof EPA and DHA esterified. In some embodiments, the composition has atleast 20% of a combination of EPA and DHA esterified. In someembodiments, the composition has at least 30% of a combination of EPAand DHA esterified. In yet other embodiments, said composition containsfrom about 5%, 10%, 20% and 30% EPA/DHA attached to position 1 and/ orposition 2. In some embodiments, the composition has a ratio of EPA/DHAranging from 1:1 to 4:1. In some embodiments, the composition has aratio of EPA/DHA ranging from 2:1 to 4:1. In some embodiments, thecomposition is acylated in a range from 60% to 80%. In some embodiments,the composition is acylated in a range from 50% to 75%.

In some embodiments, the composition further comprises a lipid carrierin a ratio of from 1:10 to 10:1 to said phospholipids. In someembodiments, the lipid carrier and said phospholipids are in a ratio offrom about 5:1 to 1:5. In some embodiments, the composition comprisesfrom about 20% to about 90% of said phospholipid composition and fromabout 10% to about 50% of said lipid carrier. The present invention isnot limited to any particular lipid carrier. In some embodiments, thelipid carrier is selected from the group consisting of a triglyceride, adiglyceride, an ethyl ester, and a methyl ester and combinationsthereof. In some embodiments, the composition provides higher uptake ofomega-3 fatty acids into plasma as compared to natural marinephospholipids when administered to subjects. In some embodiments, thecomposition improves the AA/EPA ratio in plasma phospholipids whenadministered to subjects as compared to natural marine phospholipids. Insome embodiments, the composition increases the concentration of omega-3fatty acids in tissues when administered to subjects as compared tonatural marine phospholipids. In some embodiments, the compositionreduces the concentration of biomarkers of inflammation whenadministered to subjects as compared to natural marine phospholipids. Insome embodiments, the present invention provides a food productcomprising the foregoing compositions. In some embodiments, the presentinvention provides an animal feed comprising the foregoing compositions.In some embodiments, the present invention provides a food supplementcomprising the foregoing compositions. In some embodiments, the presentinvention provides a pharmaceutical composition comprising the foregoingcompositions.

In some embodiments, the present invention provides methods of preparinga bioavailable omega-3 fatty acid composition comprising: a) providing apurified phospholipid composition comprising omega-3 fatty acid residuesand a purified triglyceride composition comprising omega-3 fatty acidresidues; b) combining said phospholipid composition and saidtriglyceride composition to form a bioavailable omega-3 fatty acidcomposition. In some embodiments, the bioavailable phospholipidcomposition is one of the compositions described above. In someembodiments, the methods further comprise the step of encapsulating saidbioavailable omega-3 fatty acid composition. In some embodiments, thebioavailable omega-3 fatty acid composition has increasedbioavailability as compared to purified triglycerides or phospholipidscomprising omega-3 fatty acid residues. In some embodiments, the methodsfurther comprise the step of packaging the bioavailable omega-3 fattyacid composition for use in functional foods. In some embodiments, themethods further comprise the step of assaying the bioavailable omega-3fatty acid composition for bioavailability. In some embodiments, themethods further comprise administering the bioavailable omega-3 fattyacid composition to a patient. In some embodiments, the presentinvention provides a food product, animal feed, food supplement orpharmaceutical composition made by the foregoing process.

In some embodiments, the present invention provides methods for reducingsymptoms of cognitive dysfunction in a child comprising administering aneffective amount of a marine phospholipid composition, wherein saidsymptoms are selected from the group consisting of ability to completetask, ability to stay on task, ability to follow instructions, abilityto complete assignments, psychomotor function, long term memory, shortterm memory, ability to make a decision, ability to follow through ondecision, ability to self-sustain attention, ability to engage inconversations, sensitivity to surroundings, ability to plan, ability tocarry out plan, ability to listen, interruptions in social situations,temper tantrums, level/frequency of frustration, level/frequencyrestlessness, frequency/level fidgeting, ability to exhibit delayedgratification, aggressiveness, demanding behavior/frequency of demandingbehavior, sleep patterns, restive sleep, interrupted sleep, awakeningbehavior, disruptive behavior, ability to exhibit control in socialsituations, ability to extrapolate information and ability to integrateinformation. In some embodiments, the child exhibits one or moresymptoms of Attention Deficit Hyperactivity Disorder (ADHD), issuspected of having ADHD, or has been diagnosed with ADHD. In someembodiments, the child exhibits one or more symptoms of autisticspectrum disorder, is suspected of having autistic spectrum disorder, orhas been diagnosed with autistic spectrum disorder. In furtherembodiments, the present invention provides methods of increasingcognitive performance in an aging mammal comprising administering aneffective amount of a marine phospholipid composition. In someembodiments, the cognitive performance is selected from the groupconsisting of memory loss, forgetfulness, short-term memory loss,aphasia, disorientation, disinhibition, and behavioral changes. In someembodiments, the mammal is a human. In some embodiments, the mammal is apet selected from the group consisting of cats and dogs. In someembodiments, the mammal has symptoms of age-associated memory impairmentor decline.

The foregoing methods are not limited to the use of any particularmarine phospholipid composition. In some embodiments, the marinephospholipid composition comprises phospholipids having the followingstructure:

wherein R1 is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is amixture of H, choline, ethanolamine, inositol and serine, saidphospholipid having at least 1% of omega-3 fatty acid moieties atpositions R1 and/or R2. In some embodiments, the phospholipidcomposition comprises from about 20-50% of OH at positions R1 and/or R2.In some embodiments, the phospholipid composition further comprises alipid carrier. In some embodiments, the phospholipid composition isprepared from natural marine phospholipids isolated from a marineorganism. In some embodiments, the phospholipid composition isenzymatically prepared by reacting lecithin with DHA and EPA in thepresence of an enzyme. In some embodiments, the lecithin is soybean oregg lecithin. In some embodiments, the omega-3 fatty acid moieties areselected from the group of EPA and DHA and combination thereof. In someembodiments, the effective amount of said phospholipid compositioncomprises from about 300 to about 1000 mg omega-3 fatty acids. In someembodiments, the phospholipid composition is administered orally. Insome embodiments, the phospholipid composition is provided in a gelcapsule or pill.

In some embodiments, the present invention provides methods of treatinga subject by administration of a marine phospholipid compositioncomprising administering a marine phospholipid composition to saidsubject under conditions such that a desired condition is improved,wherein said conditions is selected from the group consisting offertility, physical endurance, sports performance, muscle soreness,inflammation, auto-immune stimulation, metabolic syndrome, obesity andtype II diabetes. In some embodiments, the subject is a human. In someembodiments, the subject is a companion animal. The present invention isnot limited to any particular marine phospholipid composition. In someembodiments, the marine phospholipid composition comprises phospholipidshaving the following structure:

wherein R1 is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is amixture of H, choline, ethanolamine, inositol and serine, saidphospholipid having at least 1% of omega-3 fatty acid moieties atpositions R1 and/or R2. In some embodiments, the phospholipidcomposition comprises from about 20-50% of OH at positions R1 and/or R2.In some embodiments, the phospholipid composition is prepared fromnatural marine phospholipids isolated from a marine organism. In someembodiments, the composition further comprises a lipid carrier. In someembodiments, the phospholipid composition is enzymatically prepared byreacting lecithin with DHA and EPA in the presence of an enzyme. In someembodiments, the-lecithin is soybean or egg lecithin. In someembodiments, the omega-3 fatty acid moieties are selected from the groupof EPA and DHA and combination thereof. In some embodiments, theeffective amount of said phospholipid composition comprises from about300 to about 1000 mg omega-3 fatty acids. In some embodiments, thephospholipid composition is administered orally. In some embodiments,the phospholipid composition is provided in a gel capsule or pill. Insome embodiments, the human is a male.

In some embodiments, the present invention provides methods forprophylactically treating a subject by administration of a marinephospholipid composition comprising administering a marine phospholipidcomposition to a subject under conditions such that an undesirablecondition is prevented, wherein said undesirable condition is selectedfrom the group consisting of weight gain, infertility, obesity,metabolic syndrome, diabetes type II, mortality in subjects with a highrisk of sudden cardiac death, and induction of sustained ventriculartachycardia. In some embodiments, the subject is at risk for developinga condition selected from the group consisting of weight gain, obesity,metabolic syndrome, diabetes type II, mortality in subjects with a highrisk of sudden cardiac death, and induction of sustained ventriculartachycardia.

In some embodiments, the subject is a human. In some embodiments, thesubject is a companion animal. The present invention is not limited toany particular marine phospholipid composition. In some embodiments, themarine phospholipid composition comprises phospholipids having thefollowing structure:

wherein R1 is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is amixture of H, choline, ethanolamine, inositol and serine, saidphospholipid having at least 1% of omega-3 fatty acid moieties atpositions R1 and/or R2. In some embodiments, the phospholipidcomposition comprises from about 20-50% of OH at positions R1 and/or R2.In some embodiments, the phospholipid composition is prepared fromnatural marine phospholipids isolated from a marine organism. In someembodiments, the composition further comprises a lipid carrier. In someembodiments, the phospholipid composition is enzymatically prepared byreacting lecithin with DHA and EPA in the presence of an enzyme. In someembodiments, the lecithin is soybean or egg lecithin. In someembodiments, the omega-3 fatty acid moieties are selected from the groupof EPA and DHA and combination thereof. In some embodiments, theeffective amount of said phospholipid composition comprises from about300 to about 1000 mg omega-3 fatty acids. In some embodiments, thephospholipid composition is administered orally. In some embodiments,the phospholipid composition is provided in a gel capsule or pill.

DESCRIPTION OF THE FIGURES

FIG. 1. The total amount of EPA consumed during the four-week rat trial(mean±SE).

FIG. 2. Relative EPA (20:5) content of plasma (mean±SE; n=6).

FIG. 3. Relative 20:5 content of red blood cells (mean±SE; n=5-6).

FIG. 4. Relative 20:5 content of monocytes (mean±SE; n=5-6).

FIG. 5. Schematic drawing of experimental set-up.

FIG. 6. EPA levels in plasma as a function of hours after one bolusintake of a marine phospholipid composition.

FIG. 7. EPA levels in plasma as a function of hours after one bolusintake of a marine phospholipid composition.

FIG. 8. ARA levels in plasma as a function of hours after one bolusintake of a marine phospholipid composition.

DEFINITIONS

As used herein, “phospholipid” refers to an organic compound having thefollowing general structure:

wherein R1 is a fatty acid residue or —OH, R2 is a fatty acid residue or—OH, and R3 is a —H or a nitrogen containing compound such as choline(HOCH₂CH₂N⁺(CH₃)₃OH⁻), ethanolamine (HOCH₂CH₂NH₂), inositol or serine.R1 and R2 cannot simultaneously be OH. When R3 is an —OH, the compoundis a diacylglycerophosphate, while when R3 is a nitrogen-containingcompound, the compound is a phosphatide such as lecithin, cephalin,phosphatidyl serine or plasmalogen.

The R1 site is herein referred to as position 1 of the phospholipid, theR2 site is herein referred to as position 2 of the phospholipid, and theR3 site is herein referred to as position 3 of the phospholipid.

As used herein, the term omega-3 fatty acid refers to polyunsaturatedfatty acids that have the final double bond in the hydrocarbon chainbetween the third and fourth carbon atoms from the methyl end of themolecule. Non-limiting examples of omega-3 fatty acids include,5,8,11,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoicacid (DHA) and 7,10,13,16,19-docosapentanoic acid (DPA).

As used herein, the term “bioavailability” refers to the degree and rateat which a substance (as a drug) is absorbed into a living system or ismade available at the site of physiological activity.

As used herein, the term “functional food” refers to a food product towhich a biologically active supplement has been added.

As used herein, the term “fish oil” refers to any oil obtained from amarine source e.g. tuna oil, seal oil and algae oil.

As used herein, the term “lipase” refers to any enzyme capable ofhydrolyzing fatty acid esters

As used herein, the term “food supplement” refers to a food productformulated as a dietary or nutritional supplement to be used as part ofa diet.

As used herein, the term “acylation” means fatty acids attached to thephospholipid. 100% acylation means that there are no lyso- orglycerol-phospholipids.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses that the uptake/absorption of omega-3 fattyacids attached to phospholipids are dependent on the level of LPL andGPL. Preferably, in order to ensure maximum uptake the level of LPLshould be in the range of 15-45% and the level of GPL should be 0%.Furthermore, this invention discloses that the pure PC transesterifiedwith EPA/DHA have a different effect on gene expression in the liverthan 40% PC transesterified with EPA/DHA. It is disclosed that the twocompositions regulated around 40 genes differently. Furthermore, theinvention discloses that the EPA/DHA ratio is important. The treatmentcontaining a EPA/DHA ratio of 2:1 regulated key enzymes involved in theinflammatory response (NF-κB) in a positive way, the treatmentcontaining a EPA/DHA ratio of 1:1 did not.

The present invention describes novel marine lipid compositionscomprising an omega-3 containing phospholipid and a triacylglyceride(TG) in a ratio from about 1:10 to 10:1. Preferably the ratio is in therange of from about 3:1 to 1:3, more preferably the ratio is in therange of about 1:2 to 2:1. Preferably, the TG is a fish oil such as tunaoil, herring oil, menhaden oil, cod liver oil and algae oil. However,this invention is not limited to omega-3 containing oils as other TGsources are contemplated such as vegetable oils. The phospholipids inthe composition have the following structure:

wherein R1 is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is amixture of H, choline, ethanolamine, inositol and serine. Attached toposition 1 or position 2 are least 1% omega-3 fatty acids, preferably atleast 5%, more preferably at least 10% omega-3 fatty acids, up to about15%, 20%, 30%, 40%, 50%, or 60% omega-3 fatty acids. The omega-3 fattyacids can be EPA, DHA, DPA or C18:3 (n-3), most preferably the omega-3fatty acids are EPA and DHA. The phospholipid composition preferablycontains OH in position 1 or position 2 in a range of 25% to 50% inorder to maximize absorption in-vivo.

In some embodiments, the present invention provides bioavailable andbioefficient omega-3 fatty acids. This invention shows that the novelmarine lipid composition disclosed above enhances the uptake of theomega-3 fatty acid in vivo and incorporates omega-3 fatty acids moreefficiently into tissues of adult rats than pure fish oil does. Anembodiment of the invention is to use the marine lipid composition forefficient increase of omega-3 fatty acids in the liver, brain, adiposetissue, plasma, testicles and heart. Furthermore, this invention alsodiscloses that the marine lipid compositions efficiently reduced theconcentration of the pro-inflammatory precursor AA in total lipids andin phospholipids in tissues. It is disclosed that the concentration ofAA in the different lipid pools in the liver, brain, adipose tissue,plasma, testicles and heart can be more efficiently reduced than usingfish oil. Hence, the composition can be used to improve the EPA/AAratio, which is a bio-marker of silent inflammation. The invention alsodiscloses that the incorporation of the omega-3 fatty acids intomonocytes is also more efficient using the claimed marine lipidcomposition as opposed to the fish oil. Yet another embodiment of theinvention is to use the marine lipid composition to reduce chronic andacute inflammation in humans and in animals. Acute inflammation ismediated by granulocytes or polymorphonuclear leukocytes, while chronicinflammation is mediated by mononuclear cells such as monocytes.Monocytes protect against blood-borne pathogens and moves quickly tosites of infection in the tissues, secreting large amounts ofpro-inflammatory prostaglandins. Furthermore, low grade chronicinflammation may be the underlying cause of many life-style relateddiseases such as obesity, arthritis, diabetes type II, metabolicsyndrome, Alzheimer's disease, osteoarthritis, inflammatory boweldisease, allergy and asthma [14]. Hence, the marine lipid compositioncan be used to treat and prevent diseases linked to chronicinflammation. This invention discloses that the inflammatory response ofmonocytes harvested from animals in lower in animals treated with themarine lipid composition compared to fish oil. The concentrations of thepro-inflammatory cytokines such as interleukin-1β, interleukin-6 as wellas tumor necrosis factor α (TNF-α) were reduced for the group fed themarine lipid composition compared to fish oil. These cytokines areimportant markers of real inflammation as for examples I1-1β inducesfever. I1-6 also induces fever in addition to being linked to the acutephase response. TNF-α is involved in systemic inflammation as well andis released by white blood cells in the case of damage. It has a rangeof different biological effects such as increasing insulin resistance,stimulating the acute phase response in the liver and affecting thehypothalamus causing appetite suppression and fever.

This invention also discloses that the fatty acid composition of thebrain and adipose tissue phospholipids changes after in take of omega-3fatty acids for 30 days. A significant reduction of the arachidonicacids can be found in the phospholipids in the brain and adipose tissuefor the rats given either the EPA- or DHA-rich PL diets (PL 1 and PL 2,respectively). This may affect the inflammatory response in this tissueand thereby have a great impact on cognitive diseases/conditions such asParkinson's or and Alzheimer's where the inflammatory component isfundamental for the progression of the disease. This invention alsodiscloses that the reduction of ARA is present also in the sn-2 positionof the phospholipids in the brain. This is very important as thepro-inflammatory eicosanoids are produced from ARA, which arecatalytically hydrolyzed from position 2 on the phospholipid by theaction of phospholipase A2. The phospholipase A2 is released afterstimuli at the cell wall, it then moves to the nuclear membrane wherethe hydrolysis of the phospholipid takes place.

In adipose tissue, accumulation of EPA and DHA in both total lipids(table 3) and PLs (table 8) is substantial when omega-3 supplements werefed and negligible when the control diet was fed. The increase was morepronounced in total lipids, which mainly consists of triglycerides (99%of fat cell lipid content). This invention demonstrates that omega-3phospholipids can increase the accumulation of EPA/DHA into adiposetissue. This is important as the adipose tissue can function as areservoir for these fatty acids. Arachidonic acid concentration in totallipids was higher in omega-3 supplemented animals, showing probably anincrease of lipoprotein lipase activity, in agreement with the abilityof omega 3 in decreasing plasma TAGs concentration. On the other hand,arachidonic acid levels in adipose tissue PLs were significantly lowerin omega-3 supplemented animals than the levels in controls. Peculiarenough, the PL-EPA diet was the most efficient in decreasing arachidonicacid. In addition, the invention discloses that the reduction of ARA isalso observed in the sn-2 position of the phospholipids of the omega-3supplemented animals. This is very important as the pro-inflammatoryeicosanoids are produced from ARA, which are catalytically hydrolyzedfrom position 2 on the phospholipid by the action of phospholipase A2.The phospholipase A2 is released after stimuli at the cell wall, it thenmoves to the nuclear membrane where the hydrolysis of the phospholipidtakes place. The reduction of ARA in position 2 on the phospholipids mayaffect the inflammatory response in this tissue, which may havepractical application in different pathologies of the adipose tissue andin its physiological activity of accumulation and release of fattyacids.

Fatty acid data from brain are well in line with the data from adiposetissue. Also in this tissue, we found a significant decrease ofarachidonic acid in PLs, but surprisingly, only with PL-EPA and PL-DHA(table 7). On the other hand, DHA levels in both total lipids and PLswere not influenced by the omega-3 diets, while there was a small butsignificant increase in EPA levels. Lack of increase in DHA levels islikely to be attributable to the fact that the rats in this study wereadults and pass the stage in development where they incorporate DHA inthe brain (mainly PE). On the other hand, EPA being present at lowconcentration has more margin to increase. Furthermore, this may affectthe inflammatory response in this tissue, which may have a great impactin such diseases as Parkinson's and Alzheimer's where the inflammatorycomponent is fundamental for the progression of the disease. Positionaldistribution of arachidonic acid show that the ARA content is reducedfor the EPA-PL groups, as stated before this is very important as itinfluences the pro-inflammatory eicosanoid production.

In liver, as expected, we found for all omega-3 groups a significantincrease of EPA and DHA and decrease of arachidonic acid. No greatdifferences were expected between total lipids and PLs because about 80%of liver total lipids are PLs (table 4, 9 and 14).

Heart total lipids and PLs (table 6 and table 11, respectively) showed astrong increase of EPA and DHA with a concomitant decrease ofarachidonic acid when omega-3 supplements were fed. The strong decreasein the omega-6/omega-3 ratio in heart lipids is important consideringthe possible impact on the anti-inflammatory potential. Observed changein heart tissue fatty acids (increase of fatty acids with 6 or 5 doublebonds) also suggests a possible increase in membrane fluidity. Thischange was most striking in the PL-DHA group where the increase of DHAwas significantly higher than the increase in the TG-oil and PL-EPAgroups. The fluidity of myocardium cell membrane seems to play animportant role in controlling arrhythmia. Ventricular arrhythmia, is oneof the main causes of sudden cardiac death. Furthermore, atrialfibrillation is another pathological state with a high incidence andimportant health consequences.

Testicular long chain PUFAs are of special interest because there is ahigh rate of production of prostaglandins from the omega-6 PUFA(arachidonic acid mainly) into the semen or seminal fluid. High rate ofprostaglandin production does not indicate an active inflammatoryprocess but a stimulus for the uterus smooth muscle to favor malefertility. An omega-3 induced decrease of arachidonic acid as observedin other tissue could be detrimental to the male fertility if itoccurred also in testis. Furthermore, testicular tissue has also a highlevel of DPA (22:5 omega-6), which may serve as a reservoir forarachidonic acid. Arachidonic acid could be formed according to theneed, through the retroconversion mechanism in the peroxisomes. Asimilar mechanism may take place with DHA to form EPA in other tissues.Our data (table 5) show an increase of EPA and DHA and a small decreaseof arachidonic acid in the total lipids fraction when omega-3 fattyacids are fed. However, there is no change in arachidonic acid levels inPL when TG-oil and PL-EPA are fed and interestingly a significantincrease in the PL-DHA group. Furthermore, DPA n-6 concentration intotal lipids was not influenced by omega-3 supplementation but there wasa significant increase in DPA in the PL-EPA group (table 5). Overall,these data seem to indicate that the diets with omega-3 did not changethe arachidonic and DPA n-6 concentrations in a way that would predictnegative effects on male fertility. In contrast, increase in arachidonicacid content of testicular PLs (table 10) when PL-DHA was fed andincrease in DPA when PL-EPA was fed could be interpreted to bepositively associated with male fertility.

Another embodiment of the invention is to formulate the marine lipidcompositions into a feed product for the purpose of reducing low-gradechronic inflammation in animals. It can also be formulated into a foodproduct and given to humans for the same purpose. Furthermore, it can beformulated as a functional food product, as a drug or as foodsupplement.

In some embodiments, the compositions of this invention are contained inacceptable excipients and/or carriers for oral consumption. The actualform of the carrier, and thus, the compositions itself, is not critical.The carrier may be a liquid, gel, gelcap, capsule, powder, solid tablet(coated or non-coated), tea, or the like. The composition is preferablyin the form of a tablet or capsule and most preferably in the form of ahard gelatin capsule. Suitable excipient and/or carriers includemaltodextrin, calcium carbonate, dicalcium phosphate, tricalciumphosphate, microcrystalline cellulose, dextrose, rice flour, magnesiumstearate, stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethylcellulose, corn starch, and the like (includingmixtures thereof). Preferred carriers include calcium carbonate,magnesium stearate, maltodextrin, and mixtures thereof. The variousingredients and the excipient and/or carrier are mixed and formed intothe desired form using conventional techniques. The tablet or capsule ofthe present invention may be coated with an enteric coating thatdissolves at a pH of about 6.0 to 7.0. A suitable enteric coating thatdissolves in the small intestine but not in the stomach is celluloseacetate phthalate. Further details on techniques for formulation for andadministration may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

In other embodiments, the composition contains no traces of organicsolvents which is an important property regarding the safety ofconsuming such compounds. Phospholipids prepared using chemical orenzymatic methods in the presence of organic solvents may containresidual solvents that may be a health hazard. VOC are oftenco-extracted when marine phospholipids are extracted, such VOC's maycontribute to the smell taste of the phospholipids.

In other embodiments, the supplement is provided as a powder or liquidsuitable for adding by the consumer to a food or beverage. For example,in some embodiments, the dietary supplement can be administered to anindividual in the form of a powder, for instance to be used by mixinginto a beverage, or by stirring into a semi-solid food such as apudding, topping, sauce, puree, cooked cereal, or salad dressing, forinstance, or by otherwise adding to a food.

The compositions of the present invention may also be formulated with anumber of other compounds. These compounds and substances add to thepalatability or sensory perception of the particles (e.g., flavoringsand colorings) or improve the nutritional value of the particles (e.g.,minerals, vitamins, phytonutrients, antioxidants, etc.).

The dietary supplement may comprise one or more inert ingredients,especially if it is desirable to limit the number of calories added tothe diet by the dietary supplement. For example, the dietary supplementof the present invention may also contain optional ingredientsincluding, for example, herbs, vitamins, minerals, enhancers, colorants,sweeteners, flavorants, inert ingredients, and the like. For example,the dietary supplement of the present invention may contain one or moreof the following: ascorbates (ascorbic acid, mineral ascorbate salts,rose hips, acerola, and the like), dehydroepiandosterone (DHEA), Fo-Tior Ho Shu Wu (herb common to traditional Asian treatments), Cat's Claw(ancient herbal ingredient), green tea (polyphenols), inositol, kelp,dulse, bioflavinoids, maltodextrin, nettles, niacin, niacinamide,rosemary, selenium, silica (silicon dioxide, silica gel, horsetail,shavegrass, and the like), spirulina, zinc, and the like. Such optionalingredients may be either naturally occurring or concentrated forms.

In some embodiments, the dietary supplements further comprise vitaminsand minerals including, but not limited to, calcium phosphate oracetate, tribasic; potassium phosphate, dibasic; magnesium sulfate oroxide; salt (sodium chloride); potassium chloride or acetate; ascorbicacid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolonate; potassium iodide; sodium selenate; sodiummolybdate; phylloquinone; vitamin D₃; cyanocobalamin; sodium selenite;copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.Suitable dosages for vitamins and minerals may be obtained, for example,by consulting the U.S. RDA guidelines.

In further embodiments, the compositions comprise at least one foodflavoring such as acetaldehyde(ethanal), acetoin(acetyl methylcarbinol),anethole(parapropenyl anisole), benzaldehyde(benzoic aldehyde),N-butyric acid (butanoic acid), d- or l-carvone(carvol),cinnamaldehyde(cinnamic aldehyde), citral(2,6-dimethyloctadien-2,6-al-8,gera-nial, neral), decanal(N-decylaldehyde, capraldehyde, capricaldehyde, caprinaldehyde, aldehyde C-10), ethyl acetate, ethyl butyrate,3-methyl-3-phenyl glycidic acid ethylester(ethyl-methyl-phenyl-glycidate, strawberry aldehyde, C-16aldehyde), ethyl vanillin, geraniol(3,7-dimethyl-2,6 and3,6-octadien-1-ol), geranyl acetate (geraniol acetate), limonene (d-,l-, and dl-), linalool (linalol, 3,7-dimethyl-1,6-octadien-3-ol),linalyl acetate(bergamol), methyl anthranilate(methyl-2-aminobenzoate),piperonal(3,4-methylenedioxy-benzaldehyde, heliotropin), vanillin,alfalfa (Medicago sativa L.), allspice (Pimenta officinalis), ambretteseed (Hibiscus abelmoschus), angelic (Angelica archangelica), Angostura(Galipea officinalis), anise (Pimpinella anisum), star anise (Illiciumverum), balm (Melissa officinalis), basil (Ocimum basilicum), bay(Laurus nobilis), calendula (Calendula officinalis), (Anthemis nobilis),capsicum (Capsicum frutescens), caraway (Carum carvi), cardamom(Elettaria cardamomum), cassia, (Cinnamomum cassia), cayenne pepper(Capsicum frutescens), Celery seed (Apium graveolens), chervil(Anthriscus cerefolium), chives (Allium schoenoprasum), coriander(Coriandrum sativum), cumin (Cuminum cyminum), elder flowers (Sambucuscanadensis), fennel (Foeniculum vulgare), fenugreek (Trigonellafoenum-graecum), ginger (Zingiber officinale), horehound (Marrubiumvulgare), horseradish (Armoracia lapathifolia), hyssop (Hyssopusofficinalis), lavender (Lavandula officinalis), mace (Myristicafragrans), maroram (Majorana hortensis), mustard (Brassica nigra,Brassica juncea, Brassica hirta), nutmeg (Myristica fragrans), paprika(Capsicum annuum), black pepper (Piper nigrum), peppermint (Menthapiperita), poppy seed (Papayer somniferum), rosemary (Rosmarinusofficinalis), saffron (Crocus sativus), sage (Salvia officinalis),savory (Satureia hortensis, Satureia montana), sesame (Sesamum indicum),spearmint (Mentha spicata), tarragon (Artemisia dracunculus), thyme(Thymus vulgaris, Thymus serpyllum), turmeric (Curcuma longa), vanilla(Vanilla planifolia), zedoary (Curcuma zedoaria), sucrose, glucose,saccharin, sorbitol, mannitol, aspartame. Other suitable flavoring aredisclosed in such references as Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing, p. 1288-1300 (1990), and Furia andPellanca, Fenaroli's Handbook of Flavor Ingredients, The Chemical RubberCompany, Cleveland, Ohio, (1971), known to those skilled in the art.

In other embodiments, the compositions comprise at least one syntheticor natural food coloring (e.g., annatto extract, astaxanthin, beetpowder, ultramarine blue, canthaxanthin, caramel, carotenal, betacarotene, carmine, toasted cottonseed flour, ferrous gluconate, ferrouslactate, grape color extract, grape skin extract, iron oxide, fruitjuice, vegetable juice, dried algae meal, tagetes meal, carrot oil, cornendosperm oil, paprika, paprika oleoresin, riboflavin, saffron, tumeric,tumeric and oleoresin).

In still further embodiments, the compositions comprise at least onephytonutrient (e.g., soy isoflavonoids, oligomeric proanthcyanidins,indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols,ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids,conjugated fatty acids such as conjugated linoleic acid and conjugatedlinolenic acid, polyacetylene, quinones, terpenes, cathechins, gallates,and quercitin). Sources of plant phytonutrients include, but are notlimited to, soy lecithin, soy isoflavones, brown rice germ, royal jelly,bee propolis, acerola berry juice powder, Japanese green tea, grape seedextract, grape skin extract, carrot juice, bilberry, flaxseed meal, beepollen, ginkgo biloba, primrose (evening primrose oil), red clover,burdock root, dandelion, parsley, rose hips, milk thistle, ginger,Siberian ginseng, rosemary, curcumin, garlic, lycopene, grapefruit seedextract, spinach, and broccoli.

In still other embodiments, the compositions comprise at least onevitamin (e.g., vitamin A, thiamin (B1), riboflavin (B2), pyridoxine(B6), cyanocobalamin (B12), biotin, ascorbic acid (vitamin C), retinoicacid (vitamin D), vitamin E, folic acid and other folates, vitamin K,niacin, and pantothenic acid). In some embodiments, the particlescomprise at least one mineral (e.g., sodium, potassium, magnesium,calcium, phosphorus, chlorine, iron, zinc, manganese, flourine, copper,molybdenum, chromium, selenium, and iodine). In some particularlypreferred embodiments, a dosage of a plurality of particles includesvitamins or minerals in the range of the recommended daily allowance(RDA) as specified by the United States Department of Agriculture. Instill other embodiments, the particles comprise an amino acid supplementformula in which at least one amino acid is included (e.g., 1-carnitineor tryptophan).

Transesterification of phosphatidylcholine (PC) under solvent freeconditions has been performed by Haraldsson et al in 1999 [15], with theresults of high incorporation of EPA/DHA and with the followinghydrolysis profile PC/LPC/GPC=39/44/17. Extensive hydrolysis andby-product formation is generally considered a problem withtransesterification reactions, resulting in low product yields. Thisinvention discloses a process for transesterification of crude soybeanlecithin (mixture of PC, PE and PI). In the first step, the lecithin ishydrolyzed using a lipase in the presence of water (pH=8). The use of avariety of lipases is contemplated, including, but not limited to,Thermomyces Lanuginosus lipase, Rhizomucor miehei lipase, CandidaAntarctica lipase, Pseudomonas fluorescence lipase, and Mucor javanicuslipase. The first step takes around 24 hours and results in a productcomprising predominantly of lyso-phospholipids and glycerophospholipidssuch as PC/LPC/GPC=0/15/85. In the second step, free fatty acids areadded such as EPA and DHA, however any omega-3 fatty acid iscontemplated. Next a strong vacuum is applied to the reaction vessel for72 hours. However, the reaction length can be varied in order to obtaina composition with the desired amount of phospholipids andlyso-phospholipids. By extending the reaction time beyond 72 hours, aproduct comprising more than 65% phospholipids can be obtained. Next, alipid carrier is added to the reaction mixture in order to reduce theviscosity of the solution. The added amount of triglycerides can be 10%,20%, 30%, 40% or more, it depends on the requested viscosity of thefinal product. The lipid carrier can be a fish oil such as tuna oil,menhaden oil and herring oil, or any triglyceride, diglyceride, ethyl-or methylester of a fatty acid. In the final step, the product issubjected to a molecular distillation and the free fatty acids areremoved, resulting in a final product comprising of phospholipids(lyso-phospholipids and phospholipids) and triglycerides in a ratio ofpreferably 2:1.

This invention further discloses a process for the enzymatictransesterification/esterification of phospholipids with fatty acidsalkyl esters or free fatty acids in an evacuated vessel (B). A reducedpressure is applied to the vessel B (0.001-30 mbar) and water vapor(moisture) is allowed to enter the reaction mixture through a tube froma second vessel (A) (FIG. 5 for schematic drawing of the experimentalsetup). The water in vessel A is heated to 25-30° C. By adding moistureto an evacuated reaction vessel the rate of reaction could either beincreased of the lipase dosage could be reduced. In addition, the reuseof the enzymes was improved. Finally, a novel marine phospholipidcomposition was prepared characterized by being acylated in the range of55%-85%, having at least 5% EPA and/or DHA esterified, having a EPA/DHAratio of at least 1.

Accordingly, in preferred embodiments, the present invention utilizes aphospholipid, preferably a phosphatide such as lecithin. The presentinvention is not limited to the use of any particular phospholipid.Indeed, the use of a variety of phospholipids is contemplated. In someembodiments, the phospholipid is a phosphatidic or lysophosphatidicacid. In more preferred embodiments, the phospholipid is a mixture ofphosphatides such as phosphatidylcholine, phosphatidylethnolamine,phosphatidylserine and phosphatidylinositol. The present invention isnot limited to the use of any particular source of phospholipids. Insome embodiments, the phospholipids are from soybeans, while in otherembodiments, the phospholipids are from eggs. In particularly preferredembodiments, the phospholipids utilized are commercially available, suchas Alcolec 40P® from American Lecithin Company Inc (Oxford, Conn., USA).The present invention is not limited to the use of any particularenzyme. Indeed, the use of a variety of enzymes is contemplated,including, but not limited to Thermomyces Lanuginosus lipase, Rhizomucormiehei lipase, Candida Antarctica lipase, Pseudomonas fluorescencelipase, and Mucor javanicus lipase. This invention is not limited to anyparticular fatty acid alkyl ester either. This includes, but not limitedto: decanoic acid (10:0), undecanoic acid (11:0), 10-undecanoic acid(11:1), lauric acid (12:0), cis-5-dodecanoic acid (12:1), tridecanoicacid (13:0), myristic acid (14:0), myristoleic acid (cis-9-tetradecenoicacid, 14:1), pentadecanoic acid (15:0), palmitic acid (16:0),palmitoleic acid (cis-9-hexadecenoic acid, 16:1), heptadecanoic acid(17:1), stearic acid (18:0), elaidic acid (trans-9-octadecenoic acid,18:1), oleic acid (cis-9-octadecanoic acid, 18:1), nonadecanoic acid(19:0), eicosanoic acid (20:0), cis-11-eicosenoic acid (20:1),11,14-eicosadienoic acid (20:2), heneicosanoic acid (21:0), docosanoicacid (22:0), erucic acid (cis-13-docosenoic acid, 22:1), tricosanoicacid (23:0), tetracosanoic acid (24:0), nervonic acid (24:1),pentacosanoic acid (25:0), hexacosanoic acid (26:0), heptacosanoic acid(27:0), octacosanoic acid (28:0), nonacosanoic acid (29:0), triacosanoicacid (30:0), vaccenic acid (t-11-octadecenoic acid, 18:1), tariric acid(octadec-6-ynoic acid, 18:1), and ricinoleic acid(12-hydroxyoctadec-cis-9-enoic acid, 18:1) and ω3, ω6, and ω9 fatty acylresidues such as 9,12,15-octadecatrienoic acid (α-linolenic acid) [18:3,ω3]; 6,9,12,15-octadecatetraenoic acid (stearidonic acid) [18:4, ω3];11,14,17-eicosatrienoic acid (dihomo-α-linolenic acid) [20:3, ω3];8,11,14,17-eicosatetraenoic acid [20:4, ω3],5,8,11,14,17-eicosapentaenoic acid [20:5, ω3];7,10,13,16,19-docosapentaenoic acid [22:5, ω3];4,7,10,13,16,19-docosahexaenoic acid [22:6, ω3]; 9,12-octadecadienoicacid (linoleic acid) [18:2, ω6]; 6,9,12-octadecatrienoic acid(γ-linolenic acid) [18:3, ω6]; 8,11,14-eicosatrienoic acid(dihomo-γ-linolenic acid) [20:3 ω6]; 5,8,11,14-eicosatetraenoic acid(arachidonic acid) [20:4, ω6]; 7,10,13,16-docosatetraenoic acid [22:4,ω6]; 4,7,10,13,16-docosapentaenoic acid [22:5, ω6]; 6,9-octadecadienoicacid [18:2, ω9]; 8,1 1-eicosadienoic acid [20:2, ω9]; 5,8,11-eicosatrienoic acid (Mead acid) [20:3, ω9]; t10,c12 octadecadienoicacid; c10,t12 octadecadienoic acid; c9,t11 octadecadienoic acid; andt9,c11 octadecadienoic acid. Moreover, acyl residues may be conjugated,hydroxylated, epoxidated or hydroxyepoxidated acyl residues.

Marine phospholipids extracted from marine sources have a characteristicsmell and taste of rancid fish. The GC profile of the volatiles confirmsthe presence of these degradation products, such as short chainaldehydes and carboxylic acids. In preferred embodiments, the syntheticmarine phospholipid compositions of the present invention aresubstantially free of volatile organic compounds and are therefore muchmore suitable as a food supplement for humans and animals. Accordingly,in preferred compositions, the present invention provides syntheticmarine phospholipids compositions having high or increased palatability,wherein the high or increased palatability is due to low levels oforganic solvents and/or volatile organic compounds. In preferredembodiments, palatability is assayed by feeding the composition to apanel of subjects, preferably human. In more preferred embodiments, thephospholipids compositions have high or increased palatability ascompared to naturally extracted marine phospholipids. In other preferredembodiments, the synthetic marine phospholipids compositions of thepresent invention are safe for oral administration.

Experimental

EXAMPLE 1

The difference in bioavailability and bioefficacy between the marinelipid composition of the present invention and a fish oil wereinvestigated in a rat experiment. The rat feed was prepared using AIN-93except that soybean oil was removed from the feed. The pelleted AIN-93diet was ground and the marine lipid compositions (PL 1 and PL 2) aswell as fish oil (TG oil) and control were added to this ground feed.The marine lipid compositions were prepared using enzymatic (lipase)catalyzed transesterification of soy lecithin with fish oil fatty acidsaccording to the method described in Example 4, followed by the additionof a triglyceride carrier and short path distillation. The concentrationof EPA, DHA and 18:3 n-3 in the different diets can be seen in the tablebelow (table 1). TABLE 1 Amount of different fatty acid in the finalfeed products g/100 g g/100 g g/100 g SUM g/100 g EPA DHA 18:3n3 EPA +DHA + 18:3n3 Control T4 0 0 0.26 0.26 TG Oil T1 0.61 0.39 0.24 1.23 PL 1T2 0.61 0.35 0.26 1.22 PL 2 T3 0.24 0.73 0.26 1.23

Thirty six newly weaned male Sprague Dawley rats (start weight 168±11 g)were used in the experiment. The rats were initially given low-essentialoil rat feed, containing 20 g of sunflower oil and 10 g of flaxseed oilper kg of feed, for one week. After the first week, modified AIN-93 dietpowder without the test oil was given to rats ad libitum until the startof the experiment. Feeding of rats was stopped 12 hours before thesampling, 30 days after the start of feeding. Each rat was individuallyanesthetized with carbon dioxide, weighed and euthanized with cervicaldislocation. Next, blood was sampled and centrifuged to separate plasmaand blood cells. Then abdominal skin was removed and 70 ml of sterileHepes-Hanks was injected into the peritoneal cavity to collectintraperitoneal lymphocytes. The abdomen was gently massaged for about 3minutes after which the buffer solution was drained and centrifuged inFalcon tubes (200×g, 10 min) to collect the cells. The cells wereresuspended into 1 ml of freezing fluid (10% DMSO, 90% fetal bovineserum) in 1.5 ml Eppendorf tubes. These tubes were then frozen to dryice temperature for one hour by immersing the tubes in isopropanolplaced on dry ice. This enabled a slower freezing rate than by puttingthe cells directly on dry ice. In the laboratory, the cells were storedovernight at −80° C. and then stored in liquid nitrogen. Thederivatization of the lipids in order to perform gas chromatographic(GC) analysis was carried accordingly to [16]. The run conditions forthe GC were according to [17]. The growth of rats did not differ betweenthe feeding groups (data not shown). The intake of feed, and the intakefatty acids thereof, was monitored by keeping the rats in metaboliccages which allows the measurement of eaten and uneaten portion of feed.The PL 1 test group consumed somewhat less EPA than the TG oil group,whereas the PL 2 and the control group consumed much less EPA than boththe PL 1 and the TG oil groups (FIG. 1). The amount of EPA in plasmavaried between the groups and the results are shown FIG. 2. Even thoughthe estimated intake of EPA was higher in the TG Oil group than the PL 1group, the area % of EPA measured in plasma for PL1 was higher than forTG oil. Indicating a higher bioavailability of EPA from of PL 1 thanfrom the TG oil. Furthermore, this was also observed in the FA profileof the red blood cells and the monocytes (FIG. 3 and FIG. 4,respectively). Demonstrating that the PL 1 composition was moreefficient in enriching these cells with omega-3 than the TG oil group,hence being more bioefficient than TG.

EXAMPLE 2

The total fatty acid profile for the lipids in the brains (table 2),adipose tissue (table 3), liver (table 4), testicles (table 5) and heart(table 6) were isolated from the rats in example 1. The PL 1 compositionincreases the DHA content in brain and adipose tissue more than the TGcomposition. The PL 1 composition increase the EPA content in theadipose tissue more than the TG composition. It is to be observed thatthe PL1 composition increases the EPA/DHA content in the phospholipidsand in the total lipids of the different tissues as well as reduces theAA/EPA ratio more than the TG oil composition. TABLE 2 Fatty acidprofile of the total lipids isolated from the rat brain (mmol/g lipids).18:1 18:2 18:3 n3 20:3 ARA EPA 22:4 22:5 n6 22:5 n3 DHA TG-oil 370 120.35 5.7 153.7 2.2 68.5 5.6 5.0 191.4 PL-1 399 12 0.33 7.2 160.7 2.270.2 7.2 5.7 203.3 PL-2 345 13 0.33 5.7 141.6 1.7 60.8 5.8 3.4 190.3Control 363 12 0.30 5.2 174.9 0.1 81.4 7.6 1.7 189.6

TABLE 3 Fatty acid profile of the total lipids isolated from adiposetissue (mmol/g lipids). 18:1 18:2 18:3 n3 20:3 ARA EPA 22:4 22:5 n6 22:5n3 DHA TG-oil 350 306 40.2 1.4 6.3 21.7 6.3 0.9 11.3 27.5 PL-1 500 23658.2 1.9 7.0 24.0 7.0 1.0 7.2 30.1 PL-2 420 536 45.7 2.4 6.8 12.8 6.82.7 104.0 50.9 Control 419 113 28.2 0.8 3.8 0.3 3.8 0.6 26.1 0.8

TABLE 4 Fatty acid profile of the total lipids in liver (mmol/g lipids).18:1 18:2 18:3 n3 20:3 ARA EPA 22:4 22:5 n6 22:5 n3 DHA TG-oil 304.2514.1 31.9 13.2 278.9 147.8 2.5 2.9 49.9 272.8 PL-1 264.3 501.6 28.112.8 317.3 120.2 2.4 2.6 49.8 259.0 PL-2 226.6 462.7 19.9 12.3 289.080.4 3.0 5.5 30.4 263.1 Control 359.2 493.7 21.7 8.9 480.9 9.2 12.0 4.514.7 145.8

TABLE 5 Fatty acid profile of the total lipids in testicles. nmolesFA/mg n3 n6 20:3 lipids 18:4 20:5 18:3 18:3 22:6 16:1 20:4 18:2 22:520:3 n9 22:4 18:1 TG-oil 3.2 17.0 11.7 1.8 56.6 47.6 280.1 195.1 6.9285.6 3.7 56.2 326.8 PL-1 0.6 10.8 9.2 1.7 52.4 35.6 294.9 210.1 6.1294.2 3.4 61.3 348.3 PL-2 2.4 6.1 1.7 2.0 56.0 0.0 310.8 156.5 2.4 345.36.2 59.5 332.5 Control 1.8 1.1 1.6 2.0 27.2 0.0 335.5 162.2 1.3 319.15.8 78.7 330.1

TABLE 6 Fatty acid profile of the total lipids in heart. nmoles FA/mg n3n6 20:3 lipids 18:4 20:5 18:3 18:3 22:6 16:1 20:4 18:2 22:5 20:3 n9 22:418:1 TG-oil 2.0 44.7 20.2 0.9 314.0 49.7 279.6 787.9 4.0 6.8 3.0 288.9PL-1 1.5 45.0 25.6 0.8 314.4 44.6 300.4 789.0 4.4 7.2 3.3 276.6 PL-2 0.229.1 9.9 0.4 372.1 30.1 291.0 690.3 9.0 6.3 1.5 4.4 237.0 Control 0.02.9 7.2 0.7 209.9 495.8 756.2 14.7 6.2 2.4 30.3 289.5

EXAMPLE 3

The fatty acid profile of the phospholipids isolated from the brain(table 7), adipose tissue (table 8), liver (table 9), testicles (table10) and heart (Table 11) in the rats from example 1 were determined.TABLE 7 Fatty acid profile of the phospholipids isolated from the brain(mmol/g lipids). 18:1 18:2 18:3 n3 20:3 ARA EPA 22:4 22:5 n6 22:5 n3 DHATG-oil 260.8 9.9 0.2 4.5 147.17 1.9 72.8 5.6 5.3 196.8 PL-1 289.9 10.90.1 4.2 115.07 1.4 54.7 4.3 4.5 170.1 PL-2 232.6 2.1 0.6 4.2 122.38 1.257.4 4.4 0.7 175.9 Control 288.8 4.4 3.5 181.60 0.1 86.8 7.3 0.5 213.1

TABLE 8 Fatty acid profile of the phospholipids isolated from theadipose tissue (mmol/g lipids). 18:1 18:2 18:3 n3 20:3 ARA EPA 22:4 22:5n6 22:5 n3 DHA TG-oil 0.7 0.9 0.04 0.04 0.45 0.10 0.0 0.0 0.0 0.17 PL-10.6 0.5 0.04 0.02 0.24 0.06 0.0 0.0 0.0 0.09 PL-2 0.5 0.6 0.03 0.04 0.370.05 0.0 0.0 0.0 0.13 Control 0.5 0.4 0.04 0.03 0.57 0.01 0.0 0.0 0.00.04

TABLE 9 Fatty acid profile of the phospholipids isolated from the liver(mmol/g lipids). 18:1 18:2 18:3 n3 20:3 ARA EPA 22:4 22:5 n6 22:5 n3 DHATG-oil 84.2 165.4 4.4 11.5 256.9 61.6 1.7 1.69 20.5 186.5 PL-1 107.2227.6 4.6 11.4 287.3 67.9 2.0 1.62 29.1 207.7 PL-2 117.0 347.7 6.6 12.3314.7 51.7 2.7 5.12 26.0 247.7 Control 86.7 204.4 1.6 7.6 393.6 2.8 8.03.41 11.0 125.9

TABLE 10 Fatty acid profile of the phospholipids isolated from thetesticles nmoles FA/mg lipids 20:5 22:6 20:4 18:2 22:5 n3 22:5 n6 22:418:1 TG-oil 3.18 23.00 173.10 55.30 1.55 234.24 20.50 118.11 PL-1 3.7131.41 227.44 82.56 2.05 317.90 28.27 143.41 PL-2 3.339 46.79 335.87127.50 2.04 265.36 45.48 155.71 Control 0.430 14.08 204.27 49.39 0.36161.20 35.70 118.18

TABLE 11 Fatty acid profile of the phospholipids isolated from theheart. nmoles 20:3 FA/mg lipids 20:5 n3 18:3 22:6 20:4 18:2 22:5 20:3 n922:4 18:1 TG-oil 35.0 6.4 286.3 240.3 877.5 4.2 5.1 3.2 119.6 PL-1 27.56.0 261.6 205.1 754.6 3.4 4.2 2.6 96.3 PL-2 20.8 4.1 331.1 227.6 570.37.9 5.0 1.2 4.1 115.7 Control 1.6 3.1 187.1 348.8 505.9 13.7 3.2 1.922.0 128.6

TABLE 12 Fatty acid profile of the sn-2 position on the phospholipidsisolated from the adipose tissue sn-2 EPA n3 18:3 DHA ARA 18:2 TG-oil0.06 0.03 0.09 0.24 0.57 PL-1 0.09 0.04 0.12 0.29 0.70 PL-2 0.04 0.110.35 0.60 Control 0.00 0.04 0.45 0.49

TABLE 13 Fatty acid profile of the sn-2 position on the phospholipidsisolated from the brain sn-2 EPA DHA ARA 18:2 22:5 20:3 22:4 18:1 TG-oil1.6 164.7 123.6 9.8 5.0 3.9 72.8 197.4 PL-1 1.3 146.3 96.5 11.6 3.7 4.151.8 260.2 PL-2 1.2 173.5 120.3 3.2 4.4 4.2 57.4 223.4 Control 0.1 203.0171.6 4.4 7.1 3.2 81.1 258.4

TABLE 14 Fatty acid profile of the sn-2 position of the phospholipids inthe liver. n3 n6 20:3 sn-2 18:4 20:5 18:3 18:3 22:6 16:1 20:4 18:222:5n3 22:5 20:3 n9 22:4 18:1 20:2 TG-oil 0.5 50.2 2.3 0.3 145.3 6.9188.6 149.5 18.6 0.9 6.4 0.5 0.9 45.1 2.7 PL-1 0.4 60.1 2.9 0.4 176.70.0 235.6 193.0 24.3 1.2 8.3 0.8 1.6 77.9 2.5 PL-2 0.1 45.8 4.2 0.5212.4 22.2 264.5 343.7 25.6 3.7 9.6 0.6 2.0 87.0 2.5 Control 0.0 2.5 1.30.4 108.6 31.7 330.9 204.4 11.0 2.7 6.0 1.2 6.4 62.6 3.7

TABLE 15 Fatty acid profile of the sn-2 position of the phospholipids inthe testis. sn-2 20:5 22:6 20:4 18:2 22:5 22:4 18:1 TG-oil 2.9 21.8167.6 60.7 224.4 20.5 109.3 PL-1 3.4 28.4 210.7 77.2 290.6 28.2 120.3PL-2 2.5 41.6 304.7 110.4 244.1 45.4 137.8 Control 0.4 13.6 202.1 50.9157.2 35.6 113.7

TABLE 16 Fatty acid profile of the sn-2 position of the phospholipids inthe heart sn-2 20:5 n3 18:3 22:6 20:4 18:2 22:5 20:3 20:3 n9 22:4 18:1TG-oil 28.6 5.3 215.4 164.9 741.7 2.9 3.2 3.2 68.2 PL-1 22.2 4.3 193.4131.5 601.8 2.1 2.2 2.5 52.5 PL-2 17.6 3.4 256.7 160.4 519.5 5.7 3.2 1.24.1 70.0 Control 1.4 1.9 163.7 283.9 467.0 8.4 2.9 1.9 21.9 96.0

EXAMPLE 4

50 g of soy lecithin from American Lecithin Company Inc (Oxford, Conn.,USA), 40 g of TL-IM lipase from Novozymes (Bagsvaerd, Denmark) and 5 gof water (adjusted to pH=8 using NaOH) were mixed in a reaction vesselat 50° C. for 24 hours. Next, 10 g of free fatty acids containing 10%EPA and 50% DHA from Napro Pharma (Brattvaag, Norway) was added,followed by application of vacuum to the reaction vessel. After 72 hoursthe reaction was terminated and the phospholipid mixture was analyzedusing HPLC and GC. The results showed that the relationship betweenPC/LPC/GPC was 65/35/0, and that the content of EPA and DHA was around10% and 12%, respectively. Next, 20 g of sardine oil was added to thereaction mixture which comprised of 18% EPA and 12% DHA (relative GCpeak area), followed by molecular distillation. The final productcontained around 70% acetone insolubles, around 30% triglycerides andtraces of free fatty acids.

EXAMPLE 5

A marine phospholipid composition containing 8.4% EPA and 1.2% DHA wasprepared using a crude soybean lecithin as a starting material accordingto [19]. A marine oil was added to the phospholipid mixture (30% w/w) sothat the total level of EPA was 21.9% and for DHA 9.4%. Furthermore, soylecithin and lyso-phospholipids prepared according to [20] were added tothe mixture in variable amounts so that a range of PC/LPC/GPC ratioscould be obtained (Table 17). By using this method, all the treatments(MPL1-MPL5) contained exactly the same amount EPA and DHA. Lipidcompositions were consumed as a single bolus by adult Sprague-Dawleyrats and the appearance of EPA/DHA in blood was measured at differenttime points from 1 to 12 hours after ingestion. The concentration ofEPA/DHA was determined using GC-FID and reported as area percentage.FIGS. 6 and 7 show that composition MPL2 and MPL3 results in the highestconcentration of EPA/DHA in plasma after uptake. Comparing the surfacearea under each curve it is clear that MPL2 and MPL3 demonstrates ahigher bioavailability of EPA/DHA than the other composition MPL1, MPL4and MPL5. TABLE 17 Hydrolysis profile of the compositions testedTreatment MPL1 MPL2 MPL3 MPL4 MPL5 PC/LPC/GPC 85/15/0 70/30/0 55/45/040/60/0 47/37/16

EXAMPLE 6

Marine phospholipids were prepared using either 40% soy PC (AmericanLecithin Company Inc, Oxford, Conn., USA) (MPL1) or 96% pure soy PC(Phospholipid GmbH, Köln, Germany) (MPL2) according to a methoddescribed by others [4]. Fatty acid content and the level of bi-productsare shown in table 18. The MPL treatments consisted of a mixture ofphospholipids, lyso-phospholipids and glycerol-phospholipids. Lookingonly at the PC/LPC/GPC relationship, it was 64/33/2 and 42/40/18 forMPL1 and MPL2, respectively. Finally, all three treatments wereemulsified into skimmed milk. TABLE 18 Composition of the phospholipidsused in example 2 PC/LPC/ 18:2 Composition GPC (n − 6) 18:3 (n − 3) EPADHA MPL1 64/33/2  129 mg/g 9 mg/g 51 mg/g 171 mg/g MPL2 42/40/18 124mg/g 9 mg/g 96 mg/g  96 mg/g

18 newly weaned Sprague-Dawley rats were fed the milk emulsions for 1week. Each rat was placed in its own cage to ensure that they got aneven amount of test substance and the milk was consumed by the rat pupsad libitum. After 1 week the experiment was terminated and the rats weredecapitated. The animals were kept without food for 24 hours beforesampling. Entire livers were collected and frozen immediately usingliquid nitrogen (stored at −65° C.). Total RNA was isolated from theliver samples according to the Quiagen Rnaesy Midi Kit Protocol. The RNAsamples were quantified and quality measured by NanoDrop andBioanalyzer. The isolated RNA was hybridized onto a gene chip RAE230 2.0from Affymetrix (Santa Clara, Calif., USA). The expression level of eachgene was measured using an Affymetrix GeneChip 3000 7G scanner. Theresults were suitable for all chips except 2 and they were excluded fromthe trial. Using statistical tools a list of genes expresseddifferentially between MPL1 versus MPL2 (Table 3) was obtained. Theresults are based on (log) probe set summary expression measures,computed by RMA, and linear models are fitted using Empirical Bayesmethods for borrowing strength across genes (using the Limma package inR). The p-value are adjusted for multiple testing using theBenjamini-Hochberg-method, controlling the False Discovery Rate (FDR),where FDR=the proportion of null-hypotheses of no DE that are falselyrejected.

It was observed that MPL1 and MPL2 are biologically different compoundsdue to the fact that over 40 genes were differentially expressed (table19). TABLE 19 List of genes differentially expressed (DE) by MPL1 versusMPL2. The list is sorted according to increasing p-values. SLR:Estimated signal log-ratio (<0: down regulated gene, >0: up regulatedgene). Fold change: Estimated fold change corresponding to the parameter(<1: down regulated gene, >1: up regulated gene). Affy fold change:Estimated fold change using the Affymetrix definition (<−1: downregulated gene, >1: up regulated gene) df: Degrees of freedom (= numberof arrays − number of estimated parameters). Fold Affy Gene-name tp-value FDR SLR change Fold.change df Wiskott-Aldrich syndrome-like 8.350.00000 0.00261 1.02 2.02 2.02 14 Similar to osteoclast inhibitorylectin 7.40 0.00000 0.00392 1.49 2.80 2.80 14 neuron-glia-CAM-relatedcell adhesion −7.14 0.00000 0.00392 −0.55 0.68 −1.46 14 moleculedehydrodolichyl diphosphate synthase 7.09 0.00000 0.00392 0.57 1.49 1.4914 (predicted) casein kinase II, alpha 1 polypeptide 7.05 0.000000.00392 1.47 2.77 2.77 14 similar to cisplatin resistance-associated7.03 0.00000 0.00392 0.94 1.92 1.92 14 overexpressed protein (predicted)similar to Retinoblastoma-binding protein 2 7.01 0.00000 0.00392 0.671.59 1.59 14 (RBBP-2) serine/threonine kinase 25 (STE20 homolog, 6.900.00000 0.00392 0.43 1.35 1.35 14 yeast) Delangin (predicted) 6.890.00000 0.00392 0.69 1.61 1.61 14 similar to Hypothetical proteinMGC30714 −6.80 0.00000 0.00401 −0.40 0.76 −1.32 14 myeloid/lymphoid ormixed-lineage leukemia 5 6.77 0.00000 0.00401 0.68 1.60 1.60 14(trithorax homolog, Drosophila) (predicted) Radixin 6.76 0.00000 0.004010.74 1.67 1.67 14 similar to myocyte enhancer factor 2C 6.70 0.000000.00420 0.60 1.52 1.52 14 WD repeat and FYVE domain containing 1 6.640.00000 0.00443 0.80 1.74 1.74 14 (predicted) similar toRetinoblastoma-binding protein 2 6.55 0.00000 0.00509 0.87 1.82 1.82 14(RBBP-2) zinc and ring finger 1 (predicted) −6.50 0.00000 0.00528 −0.510.70 −1.42 14 pumilio 1 (Drosophila) (predicted) 6.45 0.00000 0.005350.59 1.51 1.51 14 leukocyte receptor cluster (LRC) member 8 6.44 0.000000.00535 1.06 2.08 2.08 14 (predicted) Similar to collapsin responsemediator protein- −6.40 0.00000 0.00542 −0.52 0.70 −1.43 14 2A SWI/SNFrelated, matrix associated, actin 6.37 0.00000 0.00542 0.74 1.67 1.67 14dependent regulator of chromatin, subfamily a, member 4 B-cellCLL/lymphoma 7C (predicted) 6.37 0.00000 0.00542 0.51 1.42 1.42 14synaptic vesicle glycoprotein 2b 6.33 0.00000 0.00568 0.97 1.96 1.96 14leptin receptor overlapping transcript −6.31 0.00000 0.00570 −0.58 0.67−1.50 14 Transcribed locus 6.22 0.00001 0.00658 0.44 1.36 1.36 14similar to mKIAA1321 protein 6.20 0.00001 0.00658 0.99 1.99 1.99 14retinol binding protein 2, cellular 6.18 0.00001 0.00660 0.59 1.51 1.5114 similar to Safb2 protein 6.16 0.00001 0.00660 0.62 1.53 1.53 14similar to Zbtb20 protein 6.15 0.00001 0.00660 0.49 1.40 1.40 14phosphofructokinase, liver, B-type 6.13 0.00001 0.00665 0.67 1.60 1.6014 Transcription elongation factor B (SIII), 6.10 0.00001 0.00668 0.431.35 1.35 14 polypeptide 3 Echinoderm microtubule associated proteinlike 6.10 0.00001 0.00668 1.00 2.00 2.00 14 4 (predicted) DNAtopoisomerase I, mitochondrial 6.08 0.00001 0.00678 0.61 1.53 1.53 14ectonucleoside triphosphate diphosphohydrolase 5 5.96 0.00001 0.008490.74 1.67 1.67 14 nuclear factor I/X 5.94 0.00001 0.00859 0.65 1.57 1.5714 WW domain binding protein 4 5.90 0.00001 0.00914 0.58 1.50 1.50 14Acetyl-coenzyme A acetyltransferase 1 5.84 0.00001 0.00967 0.69 1.611.61 14 similar to THO complex 2 5.84 0.00001 0.00967 0.74 1.67 1.67 14

MPL2 regulates 401 genes versus the control (table 20). A number ofgenes listed are involved maintenance of the cell, in transcription andprotein synthesis as well as signaling pathways. Others are involved inregulation of metabolism and the inflammatory response such as Tnfreceptor-associated factor 6 (Traf6_predicted) (fold change of 0.53),guanine nucleotide binding protein alpha inhibiting 2 (Gnai2) (foldchange of 0.6, gamma-butyrobetaine hydroxylase (Bbox1) (fold change of1.32), monoglyceride lipase (Mg11) (fold change 0.52), nuclear NF-kappaBactivating protein (fold change 0.65) and CCAAT/enhancer binding protein(C/EBP) (fold change of 0.66). The data listed in table 4 show that aomega-3 rich phospholipid with an EPA/DHA ratio of 2:1 behavesdifferently compared to placebo. A phospholipid composition with anEPA/DHA ratio o 1:1 did not show any difference versus placebo on geneexpression. Consequently, the EPA/DHA ratio is important and shouldpreferably be 2:1. TABLE 20 List of genes differentially expressed (DE)by MPL2 versus control. SLR: Estimated signal log-ratio (<0: downregulated gene, >0: up regulated gene). Fold change: Estimated foldchange corresponding to the parameter (<1: down regulated gene, >1: upregulated gene). Affy fold change: Estimated fold change using theAffymetrix definition (<−1: down regulated gene, >1: up regulated gene)df: Degrees of freedom (= number of arrays − number of estimatedparameters) Fold Affy Gene-ID Gene name t p-value FDR SLR changeFold.change df 1367588_a_at ribosomal protein L13A −5.22 0.00005 0.00568−0.44 0.74 −1.36 14 1367844_at guanine nucleotide binding −5.47 0.000030.00417 −0.54 0.69 −1.46 14 protein, alpha inhibiting 2 1367958_atabl-interactor 1 −6.42 0.00000 0.00119 −0.69 0.62 −1.62 14 1367971_atprotein tyrosine phosphatase −6.01 0.00001 0.00190 −0.36 0.78 −1.28 144a2 1368057_at ATP-binding cassette, sub- −5.06 0.00007 0.00688 −0.540.69 −1.45 14 family D (ALD), member 3 1368405_at v-ral simian leukemiaviral −4.86 0.00011 0.00913 −0.44 0.74 −1.36 14 oncogene homolog A (rasrelated) 1368646_at mitogen-activated protein 4.98 0.00008 0.00772 0.701.62 1.62 14 kinase 9 1368649_at dyskeratosis congenita 1, −6.73 0.000000.00080 −0.53 0.69 −1.44 14 dyskerin 1368662_at ring finger protein 39−7.01 0.00000 0.00053 −0.61 0.66 −1.52 14 1368703_at enigma homolog−5.38 0.00003 0.00450 −0.76 0.59 −1.70 14 1368824_at caldesmon 1 −7.180.00000 0.00043 −1.00 0.50 −2.00 14 1368841_at transcription factor 4−4.94 0.00009 0.00828 −0.38 0.77 −1.31 14 1368867_at GERp95 −7.830.00000 0.00019 −0.85 0.56 −1.80 14 1369094_a_at protein tyrosinephosphatase, −7.22 0.00000 0.00042 −0.97 0.51 −1.96 14 receptor type, D1369127_a_at prostaglandin F receptor 4.85 0.00011 0.00921 0.45 1.371.37 14 1369174_at SMAD, mothers against DPP −5.19 0.00005 0.00581 −0.380.77 −1.30 14 homolog 1 (Drosophila) 1369227_at Choroidermia 5.040.00007 0.00718 0.47 1.39 1.39 14 1369249_at progressive ankylosishomolog 5.36 0.00004 0.00467 0.48 1.39 1.39 14 (mouse) 1369501_at zincfinger protein 260 5.17 0.00005 0.00595 0.41 1.33 1.33 14 1369517_atpleckstrin homology, Sec7 and 4.93 0.00009 0.00829 0.48 1.40 1.40 14coiled/coil domains 1 1369546_at butyrobetaine (gamma), 2- 4.96 0.000090.00811 0.40 1.32 1.32 14 oxoglutarate dioxygenase 1(gamma-butyrobetaine hydroxylase) 1369628_at synaptic vesicleglycoprotein −7.20 0.00000 0.00042 −1.11 0.46 −2.15 14 2b 1369689_atN-ethylmaleimide sensitive 6.22 0.00001 0.00155 0.66 1.58 1.58 14 fusionprotein 1369736_at epithelial membrane protein 1 5.74 0.00002 0.002860.62 1.54 1.54 14 1369775_at nuclear ubiquitous casein −7.56 0.000000.00027 −0.79 0.58 −1.73 14 kinase and cyclin-dependent kinase substrate1370184_at cofilin 1 −6.07 0.00001 0.00178 −0.38 0.77 −1.30 141370260_at adducin 3 (gamma) −5.50 0.00003 0.00399 −0.76 0.59 −1.70 141370328_at Dickkopf homolog 3 (Xenopus 4.80 0.00012 0.00964 0.59 1.511.51 14 laevis) 1370717_at AP1 gamma subunit binding 6.00 0.000010.00192 0.58 1.50 1.50 14 protein 1 1370831_at monoglyceride lipase−5.47 0.00003 0.00414 −0.94 0.52 −1.92 14 1370901_at similar tohypothetical protein −4.83 0.00012 0.00948 −0.34 0.79 −1.27 14 MGC36831(predicted) 1370946_at nuclear factor I/X −10.64 0.00000 0.00002 −1.170.45 −2.25 14 1370949_at myristoylated alanine rich −7.58 0.000000.00026 −1.17 0.44 −2.26 14 protein kinase C substrate 1370993_atlaminin, gamma 1 6.13 0.00001 0.00171 0.63 1.54 1.54 14 1371034_at onecut domain, family −5.65 0.00002 0.00327 −1.77 0.29 −3.40 14 member 11371059_at protein kinase, cAMP- 5.24 0.00005 0.00556 0.48 1.40 1.40 14dependent, regulatory, type 2, alpha 1371345_at methyl-CpG bindingdomain −5.32 0.00004 0.00491 −0.34 0.79 −1.27 14 protein 3 (predicted)1371361_at similar to tensin −7.21 0.00000 0.00042 −0.60 0.66 −1.51 141371394_x_at similar to Ab2-143 −5.11 0.00006 0.00645 −0.63 0.64 −1.5514 1371397_at nitric oxide synthase −5.53 0.00002 0.00383 −0.34 0.79−1.26 14 interacting protein (predicted) 1371428_at −5.76 0.000010.00276 −0.37 0.77 −1.29 14 1371430_at dystroglycan 1 −5.46 0.000030.00417 −0.62 0.65 −1.53 14 1371432_at −4.95 0.00009 0.00811 −0.36 0.78−1.28 14 1371452_at bone marrow stromal cell- −5.05 0.00007 0.00705−0.46 0.73 −1.37 14 derived ubiquitin-like protein 1371573_at ribosomalprotein L36a −5.90 0.00001 0.00221 −0.40 0.76 −1.32 14 (predicted)1371589_at Ubiquitin-Like 5 Protein −5.28 0.00004 0.00518 −0.57 0.68−1.48 14 1371590_s_at Ubiquitin-Like 5 Protein −4.94 0.00009 0.00829−0.39 0.76 −1.31 14 1371779_at sorting nexin 6 (predicted) 5.64 0.000020.00329 0.63 1.55 1.55 14 1371826_at Transcribed locus −5.58 0.000020.00359 −0.48 0.72 −1.39 14 1371896_at growth arrest and DNA- −6.020.00001 0.00189 −0.43 0.74 −1.35 14 damage-inducible, gamma interactingprotein 1 (predicted) 1371918_at CD99 −5.35 0.00004 0.00476 −0.37 0.77−1.29 14 1372057_at CDNA clone MGC: 124976 −6.12 0.00001 0.00173 −0.380.77 −1.30 14 IMAGE: 7110947 1372137_at biogenesis of lysosome-related−6.03 0.00001 0.00187 −0.41 0.75 −1.32 14 organelles complex-1, subunit1 (predicted) 1372142_at arsA arsenite transporter, ATP- −4.93 0.000090.00829 −0.37 0.77 −1.30 14 binding, homolog 1 (bacterial) (predicted)1372236_at Similar to Caspase recruitment −4.90 0.00010 0.00871 −0.360.78 −1.29 14 domain protein 4 1372469_at Transcribed locus −4.840.00011 0.00945 −0.36 0.78 −1.28 14 1372697_at mitochondrial ribosomal−5.70 0.00002 0.00299 −0.58 0.67 −1.49 14 protein S15 1373031_attripartite motif protein 8 −5.13 0.00006 0.00628 −0.44 0.74 −1.36 14(predicted) 1373105_at interleukin 1 receptor-like 1 −5.01 0.000080.00742 −0.37 0.77 −1.30 14 ligand (predicted) 1373135_at similar tohypothetical protein −5.30 0.00004 0.00503 −0.55 0.68 −1.46 14 MGC27441373206_at similar to FAD104 (predicted) 6.73 0.00000 0.00080 0.64 1.561.56 14 1373303_at similar to mKIAA3013 protein −5.28 0.00004 0.00514−0.48 0.72 −1.39 14 1373347_at DMT1-associated protein −6.18 0.000010.00162 −0.73 0.60 −1.66 14 1373378_at ATP/GTP binding protein 1 5.390.00003 0.00449 0.51 1.42 1.42 14 (predicted) 1373804_at Forkhead box P1(predicted) −5.28 0.00004 0.00518 −0.59 0.66 −1.51 14 1373885_atchromobox homolog 5 −5.94 0.00001 0.00208 −1.04 0.48 −2.06 14(Drosophila HP1a) (predicted) 1374002_at −6.78 0.00000 0.00074 −0.860.55 −1.82 14 1374283_at fetal Alzheimer antigen −7.44 0.00000 0.00032−0.74 0.60 −1.67 14 (predicted) 1374425_at transducin-like enhancer of−4.91 0.00010 0.00849 −0.40 0.76 −1.32 14 split 1, homolog of DrosophilaE(spl) (predicted) 1374509_at Similar to RIKEN cDNA −5.62 0.000020.00337 −0.47 0.72 −1.39 14 1110018O08 1374511_at 5.60 0.00002 0.003450.55 1.47 1.47 14 1374657_at Transcribed locus −4.88 0.00010 0.00890−0.34 0.79 −1.27 14 1374733_at symplekin (predicted) −5.04 0.000070.00716 −0.36 0.78 −1.28 14 1374772_at similar to Chromosome 13 5.180.00005 0.00581 0.46 1.38 1.38 14 open reading frame 21 1374837_atB-cell CLL/lymphoma 7C −8.92 0.00000 0.00006 −0.71 0.61 −1.63 14(predicted) 1374851_at similar to RIKEN cDNA −4.89 0.00010 0.00879 −0.390.76 −1.31 14 2810405O22 (predicted) 1374852_at hypothetical LOC362592−5.20 0.00005 0.00579 −0.37 0.78 −1.29 14 1375214_atUDP-N-acetyl-alpha-D- −5.31 0.00004 0.00500 −0.58 0.67 −1.50 14galactosamine:polypeptide N- acetylgalactosaminyltransferase 2(predicted) 1375335_at heat shock 90 kDa protein 1, −5.26 0.000040.00538 −0.55 0.68 −1.46 14 beta 1375396_at pumilio 1 (Drosophila)−10.05 0.00000 0.00003 −0.92 0.53 −1.89 14 (predicted) 1375421_a_atpraja 2, RING-H2 motif −6.51 0.00000 0.00102 −0.60 0.66 −1.52 14containing 1375453_at −12.32 0.00000 0.00000 −1.02 0.49 −2.02 141375469_at SWI/SNF related, matrix −7.97 0.00000 0.00017 −0.93 0.53−1.90 14 associated, actin dependent regulator of chromatin, subfamilya, member 4 1375533_at vestigial like 4 (Drosophila) −5.30 0.000040.00505 −0.61 0.66 −1.52 14 (predicted) 1375548_at similar to RIKEN cDNA−5.64 0.00002 0.00328 −0.58 0.67 −1.50 14 4732418C07 (predicted)1375621_at −7.05 0.00000 0.00051 −0.96 0.51 −1.95 14 1375632_at similarto 60S ribosomal −4.85 0.00011 0.00921 −0.29 0.82 −1.22 14 protein L381375650_at bromodomain containing 4 −6.64 0.00000 0.00088 −0.48 0.71−1.40 14 (predicted) 1375658_at Transcribed locus −5.00 0.00008 0.00756−0.44 0.74 −1.35 14 1375696_at interferon (alpha and beta) 4.81 0.000120.00958 0.59 1.51 1.51 14 receptor 1 (predicted) 1375703_atmyeloid/lymphoid or mixed- −10.20 0.00000 0.00003 −1.02 0.49 −2.03 14lineage leukemia 5 (trithorax homolog, Drosophila) (predicted)1375706_at −5.01 0.00008 0.00743 −0.49 0.71 −1.40 14 1375763_at similarto 2700008B19Rik −7.08 0.00000 0.00050 −0.54 0.69 −1.45 14 protein1375958_at −5.13 0.00006 0.00628 −0.65 0.64 −1.57 14 1376059_at 5.330.00004 0.00483 0.35 1.28 1.28 14 1376256_at WD repeat and FYVE domain−9.16 0.00000 0.00005 −1.10 0.47 −2.15 14 containing 1 (predicted)1376299_at similar to Retinoblastoma- −9.22 0.00000 0.00005 −0.89 0.54−1.85 14 binding protein 2 (RBBP-2) 1376450_at transmembrane protein 5−6.26 0.00001 0.00147 −0.55 0.68 −1.46 14 (predicted) 1376523_at At richinteractive domain 4A −5.53 0.00002 0.00383 −0.77 0.59 −1.70 14 (Rbp1like) (predicted) 1376524_at hypothetical protein Dd25 −6.69 0.000000.00082 −0.66 0.63 −1.58 14 1376532_at similar to FAD104 (predicted)6.06 0.00001 0.00178 0.56 1.47 1.47 14 1376728_at Transcribed locus−4.80 0.00012 0.00966 −0.35 0.78 −1.27 14 1376917_at zinc finger protein292 −5.21 0.00005 0.00571 −0.66 0.63 −1.58 14 1376982_at Transcribedlocus −5.49 0.00003 0.00405 −0.45 0.73 −1.37 14 1377105_at −6.97 0.000000.00056 −0.89 0.54 −1.85 14 1377302_a_at methylmalonic aciduria −5.100.00006 0.00660 −0.52 0.70 −1.43 14 (cobalamin deficiency) type A(predicted) 1377524_at similar to CG18661-PA −5.36 0.00003 0.00465 −0.430.74 −1.35 14 (predicted) 1377663_at ras homolog gene family, −5.000.00008 0.00756 −0.87 0.55 −1.82 14 member E 1377683_at similar tohypothetical protein −6.63 0.00000 0.00088 −0.56 0.68 −1.47 14 FLJ13045(predicted) 1377728_at LOC499567 −5.45 0.00003 0.00419 −1.03 0.49 −2.0414 1377766_at Transcribed locus 4.80 0.00012 0.00964 0.37 1.29 1.29 141377899_at similar to RIKEN cDNA −4.99 0.00008 0.00760 −0.46 0.73 −1.3814 2810025M15 (predicted) 1377906_at DEAH (Asp-Glu-Ala-His) box −4.820.00012 0.00950 −0.73 0.60 −1.66 14 polypeptide 36 (predicted)1377914_at serine/arginine repetitive −6.41 0.00000 0.00120 −0.98 0.51−1.97 14 matrix 1 (predicted) 1378155_at similar to KIAA1096 protein−5.68 0.00002 0.00313 −0.89 0.54 −1.86 14 1378163_at Transcribed locus−4.86 0.00011 0.00913 −0.78 0.58 −1.71 14 1378170_at Transcribed locus−5.00 0.00008 0.00756 −0.92 0.53 −1.90 14 1378194_a_at rap2 interactingprotein x −4.82 0.00012 0.00950 −0.72 0.61 −1.65 14 1378361_atchromodomain helicase DNA −7.32 0.00000 0.00039 −0.73 0.60 −1.66 14binding protein 7 (predicted) 1378453_at −4.84 0.00011 0.00938 −0.740.60 −1.66 14 1378504_at Insulin-like growth factor I −5.41 0.000030.00440 −0.96 0.51 −1.95 14 mRNA, 3′ end of mRNA 1378786_at Transcribedlocus, weakly 4.89 0.00010 0.00879 0.33 1.25 1.25 14 similar toNP_780607.2 hypothetical protein LOC10905 [Mus musculus] 1379062_atsimilar to Expressed sequence −6.60 0.00000 0.00090 −1.08 0.47 −2.12 14AU019823 1379073_at Similar to RIKEN cDNA −5.51 0.00003 0.00394 −0.490.71 −1.40 14 2310067G05 1379101_at DEAH (Asp-Glu-Ala-His) box −5.550.00002 0.00375 −0.87 0.55 −1.82 14 polypeptide 36 (predicted)1379112_At At rich interactive domain 4A −5.70 0.00002 0.00299 −0.440.74 −1.35 14 (Rbp1 like) (predicted) 1379232_at TBC1D12: TBC1 domain−6.98 0.00000 0.00056 −1.40 0.38 −2.63 14 family, member 12 (predicted)1379330_s_at CDNA clone IMAGE: 7316839 −4.80 0.00012 0.00967 −0.36 0.78−1.28 14 1379332_at Transcribed locus, strongly −4.88 0.00010 0.00886−0.61 0.66 −1.52 14 similar to XP_417265.1 PREDICTED: similar to F-box-WD40 repeat protein 6 [Gallus gallus] 1379399_at similar to cDNAsequence −5.37 0.00003 0.00459 −0.42 0.75 −1.34 14 BC016188 (predicted)1379457_at neural precursor cell expressed, −5.39 0.00003 0.00449 −0.560.68 −1.48 14 developmentally down- regulated gene 1 (predicted)1379469_at similar to transducin (beta)-like −6.23 0.00001 0.00153 −0.910.53 −1.88 14 1 X-linked 1379485_at eukaryotic translation initiation−7.08 0.00000 0.00050 −1.68 0.31 −3.21 14 factor 3, subunit 10 (theta)(predicted) 1379571_at plakophilin 4 (predicted) −5.42 0.00000 0.00436−0.74 0.60 −1.67 14 1379578_at similar to Zbtb20 protein −8.89 0.000000.00006 −0.71 0.61 −1.63 14 1379662_a_at SNF related kinase 4.93 0.000090.00829 0.36 1.29 1.29 14 1379715_at similar to CG9346-PA −4.93 0.000090.00829 −0.71 0.61 −1.63 14 (predicted) 1379826_at similar tohypothetical protein −5.95 0.00001 0.00208 −0.62 0.65 −1.54 14 MGC319671380008_at similar to Neurofilament triplet −5.11 0.00006 0.00645 −0.600.66 −1.52 14 H protein (20 kDa neurofilament protein) (Neurofilamentheavy polypeptide) (NF-H) (predicted) 1380060_at DNA topoisomerase I,−5.23 0.00005 0.00566 −0.53 0.69 −1.44 14 mitochondrial 1380062_atmembrane protein, −6.88 0.00000 0.00065 −0.75 0.59 −1.68 14palmitoylated 6 (MAGUK p55 subfamily member 6) (predicted) 1380166_atSimilar to hypothetical protein 5.63 0.00002 0.00333 0.34 1.27 1.27 14FLJ12056 1380371_at delangin (predicted) −9.37 0.00000 0.00005 −0.940.52 −1.91 14 1380446_at myeloid/lymphoid or mixed- −5.00 0.000080.00756 −0.62 0.65 −1.54 14 lineage leukemia (trithorax homolog,Drosophila); translocated to, 10 (predicted) 1380503_at hypotheticalLOC305452 −6.07 0.00001 0.00178 −0.62 0.65 −1.53 14 (predicted)1380728_at Similar to collapsin response 6.09 0.00001 0.00178 0.49 1.411.41 14 mediator protein-2A 1381469_a_at PERQ amino acid rich, with−5.49 0.00003 0.00405 −0.51 0.70 −1.43 14 GYF domain 1 (predicted)1381525_at −4.82 0.00012 0.00952 −0.41 0.75 −1.33 14 1381542_at UBXdomain containing 2 −6.15 0.00001 0.00171 −0.83 0.56 −1.78 14(predicted) 1381548_at golgi phosphoprotein 4 −5.81 0.00001 0.00256−0.69 0.62 −1.61 14 (predicted) 1381567_at hypothetical LOC294390 4.970.00008 0.00800 0.36 1.29 1.29 14 (predicted) 1381764_s_at ring fingerprotein 126 −5.54 0.00002 0.00382 −0.51 0.70 −1.42 14 (predicted)1381809_at ankyrin repeat domain 11 −5.94 0.00001 0.00209 −1.11 0.46−2.17 14 (predicted) 1381829_at −6.27 0.00000 0.00145 −1.07 0.48 −2.1014 1381878_at ubinuclein 1 (predicted) −5.82 0.00001 0.00252 −1.18 0.44−2.26 14 1381958_at similar to mKIAA0259 protein −6.90 0.00000 0.00062−1.27 0.41 −2.42 14 1382000_at 4.82 0.00012 0.00950 0.41 1.33 1.33 141382009_at Transcribed locus −5.39 0.00003 0.00449 −0.69 0.62 −1.62 141382027_at LOC498010 −6.28 0.00000 0.00144 −0.76 0.59 −1.70 141382056_at similar to splicing factor p54 −8.13 0.00000 0.00016 −0.970.51 −1.96 14 1382109_at nuclear NF-kappaB activating −5.83 0.000010.00250 −0.62 0.65 −1.53 14 protein 1382155_at 6.37 0.00000 0.00126 0.581.50 1.50 14 1382193_at Transcribed locus −6.07 0.00001 0.00178 −1.420.37 −2.67 14 1382306_at Ariadne ubiquitin-conjugating 6.59 0.000000.00090 0.59 1.50 1.50 14 enzyme E2 binding protein homolog 1(Drosophila) (predicted) 1382307_at protein phosphatase 1, −4.79 0.000130.00976 −0.47 0.72 −1.39 14 regulatory (inhibitor) subunit 12A1382358_at Similar to SRY (sex −5.34 0.00004 0.00482 −0.65 0.64 −1.57 14determining region Y)-box 5 isoform a 1382372_at Aryl hydrocarbonreceptor −5.07 0.00007 0.00680 −0.74 0.60 −1.67 14 1382430_at similar toKIAA1585 protein −5.62 0.00002 0.00338 −0.58 0.67 −1.50 14 (predicted)1382434_at ectonucleoside triphosphate −5.89 0.00001 0.00229 −0.73 0.60−1.66 14 diphosphohydrolase 5 1382466_at similar to RIKEN cDNA −5.430.00003 0.00433 −0.98 0.51 −1.97 14 6530403A03 (predicted) 1382551_atsimilar to Intersectin 2 (SH3 −6.72 0.00000 0.00080 −1.41 0.38 −2.67 14domain-containing protein 1B) (SH3P18) (SH3P18-like WASP associatedprotein) 1382558_at transcription factor 3 −6.13 0.00001 0.00171 −0.620.65 −1.54 14 (predicted) 1382573_at Transcribed locus 5.08 0.000070.00677 0.38 1.30 1.30 14 1382584_at similar to mKIAA1321 protein −7.220.00000 0.00042 −1.15 0.45 −2.22 14 1382620_at ankyrin repeat domain 11−9.69 0.00000 0.00003 −0.95 0.52 −1.93 14 (predicted) 1382797_at similarto 1500019C06Rik −5.02 0.00008 0.00742 −0.47 0.72 −1.39 14 protein1382813_at similar to RIKEN cDNA −5.36 0.00004 0.00467 −0.46 0.73 −1.3814 4930444A02 (predicted) 1382862_at Transcribed locus −6.23 0.000010.00153 −1.16 0.45 −2.23 14 1382904_at similar to hypothetical protein−9.04 0.00000 0.00005 −0.85 0.56 −1.80 14 DKFZp434K1421 (predicted)1382935_at similar to Hypothetical protein −6.54 0.00000 0.00097 −0.640.64 −1.56 14 KIAA0141 1382939_at translocated promoter region −5.180.00005 0.00581 −1.13 0.46 −2.19 14 (predicted) 1382957_at similar tocisplatin resistance- −8.04 0.00000 0.00016 −1.08 0.47 −2.11 14associated overexpressed protein (predicted) 1382960_at Transcribedlocus −5.95 0.00001 0.00208 −0.77 0.59 −1.70 14 1382972_at Transcribedlocus, strongly 5.17 0.00005 0.00595 0.37 1.29 1.29 14 similar toXP_226713.2 PREDICTED: similar to Src- associated protein SAW [Rattusnorvegicus] 1383008_at SMC4 structural maintenance −5.19 0.00005 0.00581−0.98 0.51 −1.97 14 of chromosomes 4-like 1 (yeast) (predicted)1383040_a_at −5.46 0.00003 0.00419 −0.47 0.72 −1.38 14 1383052_a_at−6.54 0.00000 0.00097 −0.62 0.65 −1.53 14 1383054_at −7.86 0.000000.00019 −0.76 0.59 −1.70 14 1383060_at G kinase anchoring protein 1−5.82 0.00001 0.00255 −0.44 0.74 −1.35 14 (predicted) 1383085_at Similarto Sh3bgrl protein −5.20 0.00005 0.00576 −0.86 0.55 −1.81 14 1383179_atSimilar to hypothetical protein −5.10 0.00006 0.00660 −0.75 0.60 −1.6814 HSPC129 (predicted) 1383184_at zinc and ring finger 1 5.03 0.000070.00731 0.39 1.31 1.31 14 (predicated) 1383334_at Transcribed locus−5.37 0.00003 0.00461 −0.46 0.73 −1.37 14 1383455_atglutamyl-prolyl-tRNA −6.80 0.00000 0.00072 −0.72 0.61 −1.65 14synthetase (predicted) 1383535_at ankyrin repeat and SOCS box- 6.240.00001 0.00152 0.38 1.30 1.30 14 containing protein 8 (predicted)1383615_a_at similar to HECT domain −6.06 0.00001 0.00178 −1.08 0.47−2.11 14 containing 1 1383687_at −5.40 0.00003 0.00441 −0.43 0.74 −1.3414 1383776_at Transcribed locus 6.41 0.00000 0.00120 0.62 1.54 1.54 141383786_at Transcribed locus −5.13 0.00006 0.00628 −0.50 0.71 −1.41 141383825_at radixin −9.20 0.00000 0.00005 −1.01 0.50 −2.01 14 1383827_attousled-like kinase 1 −6.09 0.00001 0.00178 −1.25 0.42 −2.37 14(predicted) 1384125_at myeloid/lymphoid or mixed- −5.97 0.00001 0.00202−0.51 0.70 −1.42 14 lineage leukemia 5 (trithorax homolog, Drosophila)(predicted) 1384131_at ADP-ribosylation factor-like 6 6.10 0.000010.00176 0.70 1.63 1.63 14 interacting protein 2 (predicted) 1384146_atSimilar to CD69 antigen (p60, −5.22 0.00005 0.00568 −1.35 0.39 −2.55 14early T-cell activation antigen) 1384154_at WW domain binding protein 4−5.33 0.00004 0.00483 −0.52 0.70 −1.43 14 1384260_at Transcribed locus−6.36 0.00000 0.00126 −0.66 0.63 −1.58 14 1384263_at ATP-bindingcassette, sub- −7.27 0.00000 0.00040 −0.72 0.61 −1.64 14 family A(ABC1), member 13 (predicted) similar to hypothetical protein MGC33214(predicted) 1384339_s_at casein kinase II, alpha 1 −8.81 0.00000 0.00006−1.83 0.28 −3.56 14 polypeptide 1384376_at similar to FLJ14281 protein−5.42 0.00003 0.00436 −0.65 0.64 −1.57 14 1384394_at −7.21 0.000000.00042 −0.61 0.65 −1.53 14 1384609_a_at similar to RIKEN cDNA −6.610.00000 0.00090 −0.91 0.53 −1.87 14 B230380D07 (predicted) 1384766_a_atsimilar to PHD finger protein −5.18 0.00005 −0.00581 −0.70 0.61 −1.63 1414 isoform 1 1384791_at UDP-GlcNAc:betaGal beta- −5.08 0.00006 0.00671−0.75 0.60 −1.68 14 1,3-N- acetylglucosaminyltransferase 1 (predicted)1384792_at formin binding protein 3 −6.70 0.00000 0.00082 −0.97 0.51−1.96 14 (predicted) 1384857_at A kinase (PRKA) anchor −5.62 0.000020.00338 −1.05 0.48 −2.07 14 protein (yotiao) 9 1385006_at alphathalassemia/mental −4.94 0.00009 0.00829 −0.45 0.73 −1.37 14 retardationsyndrome X-linked homolog (human) 1385038_at similar tohedgehog-interacting −9.94 0.00000 0.00003 −0.80 0.57 −1.75 14 protein1385076_at −5.78 0.00001 0.00271 −0.57 0.68 −1.48 14 1385077_at similarto golgi-specific −7.83 0.00000 0.00019 −1.05 0.48 −2.07 14 brefeldinA-resistance guanine nucleotide exchange factor 1 (predicted)1385101_a_at Unknown (protein for −5.53 0.00002 0.00383 −0.97 0.51 −1.9614 MGC: 73017) 1385108_at Transcribed locus −4.83 0.00011 0.00946 −1.270.42 −2.40 14 1385240_at WD repeat domain 33 −4.81 0.00012 0.00958 −0.930.52 −1.91 14 (predicted) 1385320_at similar to Pdz-containing −5.260.00004 0.00530 −0.47 0.72 −1.38 14 protein 1385407_at TCDD-induciblepoly(ADP- −5.46 0.00003 0.00417 −1.34 0.39 −2.54 14 ribose) polymerase(predicted) 1385408_at similar to mKIAA0518 protein −5.86 0.000010.00236 −1.31 0.40 −2.49 14 1385689_at Transcribed locus −4.83 0.000110.00948 −0.68 0.62 −1.60 14 1385852_at CREB binding protein −4.850.00011 0.00927 −0.53 0.69 −1.44 14 hypothetical gene supported byNM_133381 1385931_at hook homolog 3 −7.30 0.00000 0.00040 −1.66 0.32−3.16 14 1385999_at YME1-like 1 (S. cerevisiae) −4.80 0.00012 0.00964−0.63 0.64 −1.55 14 1386191_a_at Transcribed locus 5.22 0.00005 0.005680.46 1.37 1.37 14 1386641_at Transcribed locus −5.41 0.00003 0.00440−0.97 0.51 −1.95 14 1386793_at similar to zinc finger protein 61 −5.280.00004 0.00514 −0.59 0.66 −1.51 14 1387087_at CCAAT/enhancer binding−5.43 0.00003 0.00435 −0.61 0.66 −1.52 14 protein (C/EBP), beta1387306_a_at early growth response 2 4.82 0.00012 0.00950 0.33 1.26 1.2614 1387365_at nuclear receptor subfamily 1, −4.86 0.00011 0.00913 −0.350.78 −1.27 14 group H, member 3 1387415_a_at syntaxin binding protein 54.86 0.00011 0.00913 0.40 1.32 1.32 14 (tomosyn) 1387458_at ring fingerprotein 4 7.05 0.00000 0.00051 0.75 1.69 1.69 14 1387664_at ATPase, H+transporting, V1 5.55 0.00002 0.00375 0.46 1.38 1.38 14 subunit B,isoform 2 1387757_at liver regeneration p-53 related 5.19 0.000050.00581 0.51 1.42 1.42 14 protein 1387760_a_at one cut domain, family−6.12 0.00001 0.00171 −1.58 0.34 −2.98 14 member 1 1387789_at v-etserythroblastosis virus E26 −6.61 0.00000 0.00090 −0.58 0.67 −1.50 14oncogene like (avian) 1387915_at Ratsg2 −4.79 0.00013 0.00976 −0.33 0.79−1.26 14 1387947_at v-maf musculoaponeurotic −5.08 0.00007 0.00674 −0.800.57 −1.74 14 fibrosarcoma oncogene family, protein B (avian)1388022_a_at dynamin 1-like 4.95 0.00009 0.00811 0.43 1.35 1.35 141388059_a_at solute carrier family 11 5.75 0.00001 0.00280 0.43 1.351.35 14 (proton-coupled divalent metal ion transporters), member 21388089_a_at ring finger protein 4 5.73 0.00002 0.00289 0.50 1.41 1.4114 1388157_at myristoylated alanine rich −5.76 0.00001 0.00276 −0.510.70 −1.43 14 protein kinase C substrate 1388196_at NCK-associatedprotein 1 5.31 0.00004 0.00500 0.48 1.40 1.40 14 1388251_at proteinkinase C, lambda 5.21 0.00005 0.00571 0.55 1.47 1.47 14 1388313_atribosomal protein s25 −4.83 0.00011 0.00946 −0.63 0.65 −1.54 141388353_at proliferation-associated 2G4, −6.35 0.00000 0.00127 −0.670.63 −1.59 14 38 kDa 1388388_at Protein phosphatase 2, −5.32 0.000040.00487 −0.41 0.75 −1.33 14 regulatory subunit B (B56), delta isoform(predicted) 1388396_at serine/threonine kinase 25 −5.49 0.00003 0.00404−0.34 0.79 −1.27 14 (STE20 homolog, yeast) 1388503_at similar toCREBBP/EP300 −6.06 0.00001 0.00178 −0.40 0.76 −1.32 14 inhibitoryprotein 1 1388714_at elongation factor RNA −5.88 0.00001 0.00229 −0.460.73 −1.37 14 polymerase II (predicted) 1388735_at Similar to keratinassociated 4.84 0.00011 0.00945 0.50 1.41 1.41 14 protein 10-61388752_at BCL2-associated transcription −4.81 0.00012 0.00958 −0.400.76 −1.32 14 factor 1 (predicted) 1388849_at Protease, serine, 25(predicted) −5.97 0.00001 0.00202 −0.50 0.71 −1.41 14 1388888_atTranscribed locus 5.13 0.00006 0.00632 0.40 1.32 1.32 14 1389268_atsimilar to DNA polymerase −5.21 0.00005 0.00571 −0.37 0.77 −1.29 14lambda 1389307_at similar to Amyloid beta (A4) −4.91 0.00010 0.00854−0.49 0.71 −1.40 14 precursor-like protein 1 1389419_at Transcribedlocus −6.54 0.00000 0.00097 −1.30 0.40 −2.47 14 1389432_at pre-B-cellleukemia −4.93 0.00009 0.00829 −0.55 0.69 −1.46 14 transcription factor2 1389444_at Transcribed locus −6.84 0.00000 0.00068 −1.06 0.48 −2.09 141389806_at Transcribed locus −7.63 0.00000 0.00026 −0.54 0.69 −1.46 141389868_at similar to RCK −6.34 0.00000 0.00128 −1.47 0.36 −2.76 141389963_at P55 mRNA for p55 protein −5.54 0.00002 0.00378 −0.44 0.74−1.35 14 1389986_at LOC499304 −5.39 0.00003 0.00449 −2.57 0.17 −5.93 141389989_at alpha thalassemia/mental −4.93 0.00009 0.00829 −0.54 0.69−1.45 14 retardation syndrome X-linked homolog (human) 1389998_atNuclear receptor subfamily 2, −6.03 0.00001 0.00187 −0.69 0.62 −1.61 14group F, member 2 1390048_at serine/arginine repetitive −5.81 0.000010.00256 −1.04 0.49 −2.05 14 matrix 2 (predicted) 1390120_a_at ringfinger protein 1 −5.70 0.00002 0.00299 −0.36 0.78 −1.28 14 1390121_atGLIS family zinc finger 2 4.81 0.00012 0.00958 0.43 1.34 1.34 14(predicted) 1390227_at CDNA clone IMAGE: 7300848 −5.91 0.00001 0.00219−1.03 0.49 −2.04 14 1390360_a_at similar to Safb2 protein −4.79 0.000130.00976 −0.48 0.72 −1.39 14 1390410_at Transcribed locus −4.79 0.000120.00973 −0.49 0.71 −1.40 14 1390436_at Autophagy 7-like (S. cerevisiae)−7.88 0.00000 0.00019 −1.46 0.36 −2.76 14 (predicted) 1390448_at similarto 1110065L07Rik 5.08 0.00007 0.00674 0.32 1.25 1.25 14 protein(predicted) 1390454_at 4-nitrophenylphosphatase −5.47 0.00003 0.00415−0.41 0.75 −1.33 14 domain and non-neuronal SNAP25-like protein homolog1 (C. elegans) (predicted) 1390576_at Transcribed locus −5.10 0.000060.00660 −0.67 0.63 −1.59 14 1390660_at T-box 2 (predicted) 5.01 0.000080.00752 0.40 1.32 1.32 14 1390706_at spectrin beta 2 −5.55 0.000020.00376 −0.71 0.61 −1.64 14 1390739_at similar to zinc finger protein−5.51 0.00003 0.00395 −0.52 0.70 −1.43 14 609 similar to zinc fingerprotein 609 1390777_at sterol-C5-desaturase (fungal −6.59 0.000000.00090 −0.70 0.61 −1.63 14 ERG3, delta-5-desaturase) homolog (S.cerevisae) 1390779_at Similar to phosphoseryl-tRNA −4.86 0.00011 0.00913−0.64 0.64 −1.56 14 kinase 1390813_at Similar to RNA-binding −5.190.00005 0.00581 −0.62 0.65 −1.54 14 protein Musashi2-S 1390884_a_atUDP-GlcNAc: betaGal beta- 4.87 0.00010 0.00904 0.49 1.40 1.40 14 1,3-N-acetylglucosaminyltransferase 7 (predicted) 1391021_at similar toKIAA1749 protein −7.55 0.00000 0.00027 −0.74 0.60 −1.67 14 (predicted)1391170_at similar to mKIAA1757 protein −9.21 0.00000 0.00005 −2.01 0.25−4.04 14 (predicted) 1391222_at similar to Nedd4 binding −5.91 0.000010.00219 −0.81 0.57 −1.76 14 protein 1 (predicted) 1391297_at RESTcorepressor 1 (predicted) −5.17 0.00005 0.00595 −0.92 0.53 −1.89 141391578_at −8.48 0.00000 0.00009 −1.11 0.46 −2.15 14 1391584_atTranscribed locus 6.04 0.00001 0.00185 0.45 1.37 1.37 14 1391625_atWiskott-Aldrich syndrome-like −10.52 0.00000 0.00002 −1.28 0.41 −2.43 14(human) 1391669_at protein tyrosine phosphatase, −6.21 0.00001 0.00156−0.82 0.56 −1.77 14 receptor type, B (predicted) 1391689_at similar toRetinoblastoma- −9.06 0.00000 0.00005 −1.20 0.44 −2.30 14 bindingprotein 2 (RBBP-2) 1391701_at MYST histone −5.18 0.00005 0.00581 −0.980.51 −1.97 14 acetyltransferase (monocytic leukemia) 3 (predicted)1391743_at ELAV (embryonic lethal, −4.80 0.00012 0.00964 −1.36 0.39−2.58 14 abnormal vision, Drosophila)- like 1 (Hu antigen R) (predicted)1391830_at copine VIII (predicted) −5.22 0.00005 0.00568 −1.08 0.47−2.12 14 1391838_at ankyrin repeat domain 11 −7.81 0.00000 0.00019 −1.150.45 −2.23 14 (predicted) 1391848_at RNA binding motif protein 27 −7.020.00000 0.00053 −0.76 0.59 −1.70 14 (predicted) 1391968_at Similar toexpressed sequence −4.89 0.00010 0.00883 −0.69 0.62 −1.62 14 AA4158171392000_at Similar to PHD finger protein 5.01 0.00008 0.00742 0.45 1.371.37 14 14 isoform 1 1392061_at minichromosome maintenance 5.34 0.000040.00482 0.54 1.46 1.46 14 deficient 10 (S. cerevisiae) (predicted)1392269_at transcriptional regulator, −6.23 0.00001 0.00153 −1.13 0.46−2.19 14 SIN3A (yeast) (predicted) 1392277_at −7.29 0.00000 0.00040−0.48 0.72 −1.40 14 1392322_at GTPase, IMAP family member 7 −4.830.00012 0.00948 −0.29 0.82 −1.22 14 1392472_at similar to myocyteenhancer −9.77 0.00000 0.00003 −0.88 0.54 −1.84 14 factor 2C 1392552_atsimilar to transcription −6.15 0.00001 0.00169 −0.96 0.51 −1.95 14repressor p66 (predicted) 1392564_at myeloid/lymphoid or mixed- −6.130.00001 0.00171 −0.57 0.68 −1.48 14 lineage leukemia 5 (trithoraxhomolog, Drosophila) (predicted) 1392629_a_at similar to MADP-1 protein−4.93 0.00009 0.00829 −0.82 0.57 −1.77 14 (predicted) 1392738_at similarto KIAA1096 protein −5.88 0.00001 0.00231 −0.75 0.59 −1.68 14 1392825_atLOC499256 −5.20 0.00005 0.00580 −0.93 0.53 −1.90 14 1392864_at RhoGTPase activating protein −8.05 0.00000 0.00016 −1.37 0.39 −2.58 14 5(predicted) 1392932_at leukocyte receptor cluster −4.81 0.00012 0.00958−0.79 0.58 −1.73 14 (LRC) member 8 (predicted) 1392936_at similar to RNAbinding motif −4.82 0.00012 0.00950 −0.88 0.54 −1.85 14 protein 251392984_at copine III (predicted) −7.83 0.00000 0.00019 −0.95 0.52 −1.9314 1393151_at 5.03 0.00007 0.00726 0.65 1.57 1.57 14 1393226_atTranscribed locus −4.94 0.00009 0.00828 −0.73 0.60 −1.66 14 1393290_atsimilar to myocyte enhancer −5.65 0.00002 0.00327 −0.50 0.71 −1.42 14factor 2C 1393322_at TAF15 RNA polymerase II, −6.18 0.00001 0.00162−1.00 0.50 −2.00 14 TATA box binding protein (TBP)-associated factor(predicted) 1393378_at −5.72 0.00002 0.00293 −0.52 0.70 −1.43 141393443_a_at similar to CGI-112 protein −5.33 0.00004 0.00483 −0.47 0.72−1.39 14 (predicted) 1393505_x_at similar to RIKEN cDNA −7.60 0.000000.00026 −0.69 0.62 −1.61 14 B230380D07 (predicted) 1393511_at similar togalactose-3-O- 5.10 0.00006 0.00655 0.41 1.33 1.33 14 sulfotransferase 41393560_at −4.91 0.00010 0.00852 −0.51 0.70 −1.42 14 1393576_atTranscribed locus −4.82 0.00012 0.00950 −0.62 0.65 −1.54 14 1393593_atsimilar to KIAA0597 protein 5.43 0.00003 0.00435 0.57 1.48 1.48 141393639_at myosin X (predicted) −4.95 0.00009 0.00811 −0.59 0.67 −1.5014 1393790_at HRAS-like suppressor 5.44 0.00003 0.00432 0.44 1.35 1.3514 (predicted) 1393798_at alpha thalassemia/mental −5.00 0.00008 0.00757−0.84 0.56 −1.79 14 retardation syndrome X-linked homolog (human)1393804_at similar to hypothetical protein −6.79 0.00000 0.00073 −0.850.56 −1.80 14 FLJ22490 (predicted) 1393809_at Tnf receptor-associatedfactor 6 −8.48 0.00000 0.00009 −0.90 0.53 −1.87 14 (predicted)1393811_at similar to putative repair and −6.08 0.00001 0.00178 −0.790.58 −1.73 14 recombination helicase RAD26L 1393910_at similar toFam13a1 protein −4.85 0.00011 0.00921 −0.81 0.57 −1.75 14 (predicted)1393981_at similar to KIAA0423 −5.24 0.00005 0.00556 −0.57 0.68 −1.48 14(predicted) 1394003_at similar to DNA polymerase −5.59 0.00002 0.00349−0.59 0.67 −1.50 14 epsilon p17 subunit (DNA polymerase epsilon subunit3) (Chromatin accessibility complex 17) (HuCHRAC17) (CHRAC-17)1394220_at Similar to hypothetical protein 5.46 0.00003 0.00417 0.431.34 1.34 14 (predicted) 1394243_at similar to spermine synthase −6.110.00001 0.00175 −0.60 0.66 −1.51 14 1394436_at sperm associated antigen9 −6.60 0.00000 0.00090 −0.91 0.53 −1.88 14 (predicted) 1394497_atsimilar to TCF7L2 protein −8.03 0.00000 0.00016 −1.06 0.48 −2.08 141394594_at Transcribed locus 5.09 0.00006 0.00671 0.42 1.34 1.34 141394715_at Dicer1, Dcr-1 homolog 5.14 0.00006 0.00627 0.54 1.46 1.46 14(Drosophila) (predicted) 1394740_at 5.41 0.00003 0.00440 0.52 1.43 1.4314 1394742_at Transcribed locus −5.73 0.00002 0.00289 −0.98 0.51 −1.9814 1394746_at hect (homologous to the E6-AP −7.32 0.00000 0.00039 −0.940.52 −1.91 14 (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-likedomain (RLD) 1 (predicted) 1394814_at translocated promoter region −6.130.00001 0.00171 −0.63 0.64 −1.55 14 (predicted) 1394849_at Transcribedlocus −5.22 0.00005 0.00569 −1.61 0.33 −3.05 14 1394865_at Transmembraneprotein 7 −7.85 0.00000 0.00019 −0.92 0.53 −1.90 14 (predicted)1394965_at enthoprotin 5.30 0.00004 0.00503 0.40 1.32 1.32 14 1394969_atTranscribed locus 5.40 0.00003 0.00441 0.39 1.31 1.31 14 1394985_atearly endosome antigen 1 −7.60 0.00000 0.00026 −1.00 0.50 −2.00 14(predicted) 1395211_s_at supervillin (predicted) −8.74 0.00000 0.00007−0.98 0.51 −1.97 14 1395237_at eukaryotic translation initiation −8.310.00000 0.00012 −0.87 0.55 −1.83 14 factor 5B 1395264_at similar toRap1-interacting −6.85 0.00000 0.00067 −0.95 0.52 −1.93 14 factor 11395331_at similar to hypothetical protein 4.84 0.00011 0.00945 0.311.24 1.24 14 CL25084 (predicted) 1395338_at leucine-rich PPR-motif 5.240.00005 0.00555 0.75 1.68 1.68 14 containing (predicted) 1395516_atsimilar to hypothetical protein −4.89 0.00010 0.00883 −0.59 0.66 −1.5114 FLJ10154 (predicted) 1395565_at COP9 signalosome subunit 4 5.550.00002 0.00376 0.40 1.32 1.32 14 1395610_at similar to Hypotheticalprotein 5.66 0.00002 0.00325 0.33 1.26 1.26 14 MGC30714 1395616_atsimilar to Ab2-008 (predicted) −5.03 0.00007 0.00729 −0.50 0.71 −1.42 141395625_at Transcribed locus −6.03 0.00001 0.00187 −0.76 0.59 −1.70 141395739_at similar to RIKEN cDNA 5.05 0.00007 0.00698 0.54 1.46 1.46 14C920006C10 (predicted) 1395814_at Transcribed locus −5.09 0.000060.00663 −0.78 0.58 −1.71 14 1395976_at similar to phosphoinositol 4-−6.37 0.00000 0.00126 −0.57 0.67 −1.49 14 phosphate adaptor protein-21395981_at helicase, ATP binding 1 −5.76 0.00001 0.00276 −0.62 0.65−1.54 14 (predicted) 1396036_at Ral GEF with PH domain and −6.67 0.000000.00084 −1.04 0.49 −2.06 14 SH3 binding motif 2 (predicted) 1396063_atDEK oncogene (DNA binding) −4.82 0.00012 0.00952 −0.63 0.65 −1.55 141396100_at similar to RIKEN cDNA −5.15 0.00006 0.00610 −0.56 0.68 −1.4714 2010009L17 (predicted) 1396170_at WW domain binding protein 4 −7.780.00000 0.00020 −0.77 0.59 −1.71 14 1396187_at Hypothetical protein 5.140.00006 0.00622 0.51 1.43 1.43 14 LOC606294 1396202_at Transcribed locus4.97 0.00008 0.00795 0.52 1.44 1.44 14 1396403_at −9.07 0.00000 0.00005−1.01 0.50 −2.02 14 1396803_at similar to THO complex 2 −7.09 0.000000.00050 −0.90 0.54 −1.86 14 1397203_at PRP4 pre-mRNA processing −6.180.00001 0.00162 −0.67 0.63 −1.59 14 factor 4 homolog B (yeast)(predicted) 1397234_at G patch domain containing 1 −5.65 0.00002 0.00326−0.49 0.71 −1.40 14 (predicted) 1397367_at A disintegrin and 5.050.00007 0.00698 0.47 1.38 1.38 14 metalloprotease domain 23 (predicted)1397508_at similar to RIKEN cDNA −5.08 0.00006 0.00671 −0.62 0.65 −1.5414 2310005B10 1397552_at echinoderm microtubule −8.47 0.00000 0.00009−1.39 0.38 −2.62 14 associated protein like 4 (predicted) 1397627_atdiaphanous homolog 1 −5.07 0.00007 0.00680 −0.52 0.70 −1.43 14(Drosophila) (predicted) 1397647_at solute carrier family 25 5.510.00003 0.00395 0.62 1.54 1.54 14 (mitochondrial carrier; ornithinetransporter) member 15 (predicted) 1397669_at Chemokine (C—C motif) 5.780.00001 0.00271 0.51 1.43 1.43 14 receptor 6 (predicted) 1397674_ateukaryotic translation initiation −6.44 0.00000 0.00116 −0.76 0.59 −1.6914 factor 3, subunit 8, 110 kDa (predicted) 1397676_at Similar toosteoclast inhibitory −6.68 0.00000 0.00084 −1.34 0.39 −2.54 14 lectin1397758_at Similar to choline −4.83 0.00011 0.00946 −0.38 0.77 −1.30 14phosphotransferase 1; cholinephosphotransferase 1 alpha;cholinephosphotransferase 1 1397959_at similar to RIKEN cDNA −6.390.00000 0.00123 −1.14 0.45 −2.20 14 D130059P03 gene (predicted)1398311_a_at kinase D-interacting substance 5.14 0.00006 0.00627 0.441.36 1.36 14 220 1398351_at Ubiquitin specific protease 7 −5.60 0.000020.00349 −0.42 0.75 −1.34 14 (herpes virus-associated) (predicted)1398420_at Similar to E3 ubiquitin ligase −5.33 0.00004 0.00483 −0.940.52 −1.92 14 SMURF2 (predicted) 1398436_at ubiquitin specific protease42 −6.36 0.00000 0.00126 −0.76 0.59 −1.69 14 (predicted) 1398486_at CDNAclone MGC: 93990 −8.09 0.00000 0.00016 −1.53 0.35 −2.89 14 IMAGE:7115381 1398522_at similar to Ab2-034 (predicted) −4.92 0.00009 0.00832−0.51 0.70 −1.42 14 1398553_at similar to CGI-100-like protein −6.910.00000 0.00062 −1.68 0.31 −3.20 14 1398834_at mitogen activated protein−4.94 0.00009 0.00828 −0.32 0.80 −1.25 14 kinase kinase 2 1398926_atprefoldin 1 (predicted) −5.95 0.00001 0.00208 −0.48 0.72 −1.40 141398963_at TAF10 RNA polymerase II, −5.42 0.00003 0.00436 −0.41 0.75−1.33 14 TATA box binding protein (TBP)-associated factor (predicted)1399099_at heterogeneous nuclear −4.94 0.00009 0.00829 −0.54 0.69 −1.4614 ribonucleoprotein U-like 1 (predicted) 1399140_at Transcribed locus−5.16 0.00005 0.00597 −0.49 0.71 −1.40 14 AFFX-BioB- Biotin synthase−4.89 0.00010 0.00879 −0.64 0.64 −1.56 14 M_at biotin synthesis, sulfurinsertion? AFFX- dethiobiotin synthetase −4.92 0.00009 0.00834 −0.700.62 −1.62 14 BioDn-5_at AFFX-r2-Ec- dethiobiotin synthetase −5.410.00003 0.00440 −0.51 0.70 −1.43 14 bioD-5_at

EXAMPLE 7

60 g fatty acid ethyl ester consisting of 10% EPA and 50% DHA (FAEE10-50), obtained from Napro Pharma (Brattvaag, Norway) and 15 g TL-IMobtained from Novozymes (Bagsvaerd, Denmark) were mixed in an evacuatedround bottomed glass flask for 15 minutes. Next, N₂ was released intothe glass flask and the mixture was heated to 65° C. 20 g Alcolec 40P®from American Lecithin Company Inc (Oxford, Conn., USA) was then addedto the reaction mixture. Alcolec 40P® is a crude soybean phospholipidproduct containing 40% PC, 26% phosphatidylethanolamine, 11%phosphatidylinositol, 1% phosphatidylserine, 13% phytoglycolipids aswell as 14% other phosphatides (w,w). Next, the glass flask wasevacuated (20-30 mbar). Finally, a second vessel containing water (30°C.), was connected to the reaction vessel through a plastic tube (FIG.1). The reduced pressure allowed moisture from the headspace of thesecond vessel to be added through the reaction mixture continuously. Inorder to obtain the final product the enzymes were removed byfiltration. Finally, a triglyceride carrier was added to the product,followed by removal of the residual free fatty acids and/or esters byshort path distillation. In order to analyze the product, the sample wasfractionated by HPLC-UV (λ=207 nm) with a silica column andmethanol-water (92:8, v/v) as mobile phase. The isolated PC+LPC fractionwas then dried under nitrogen prior to derivatization; finally the fattyacid profile was determined by analyzing the derivatives using GC-FID.Furthermore, the relationship between PC, LPC and GPC was determinedusing HPLC with the method above, except that the UV detector wasreplaced by an evaporative light scattering detection (ELSD). IntegratedELSD peak areas were reported for PC/LPC/GPC (total 100%); however forsimplicity other PL species were not analyzed. The results obtained forexample 7 is shown in table 20 below. TABLE 20 Results obtained aftertransesterification using vacuum and water addition Reaction timePC/LPC/GPC* EPA/DHA** Acid value 1 day 65/31/4 3.6/2.6 43 2 days 52/45/35.3/4.7 55 5 days 78/22/0 5.2/4.6 65 6 days 72/26/2 6.0/5.3 75*ELSD peak area (total 100%). Only peaks relating to PC, LPC and GPC areintegrated.**EPA/DHA attached to PC + LPC

EXAMPLE 8

The enzymes from example 7 were isolated by filtration and thepossibility of reuse was determined in the following experiment. 30 gFAEE (10-50), 10 g Alcolec and 15 g used enzymes (equivalent to 7.5 genzyme because the used enzymes had absorbed product from the firstreaction). The reaction was performed at 65° C. and stirred at 200 rpmusing a shaker incubator. The transesterified phospholipids wereanalyzed as in the previous example and the results are shown in table21 below. TABLE 21 Results obtained with reused enzymes using incubatorshaker Reaction time PC/LPC/GPC* EPA/DHA** Acid value 1 day 94/5/10.4/0.5 55 2 days 87/11/2 0.7/0.7 76 5 days 68/26/6 1.0/0.8 85*ELSD peak area (total 100%). Only peaks relating to PC, LPC and GPC areintegrated.**EPA/DHA attached to PC + LPC

EXAMPLE 9

The same conditions as in example 1 were used, except that the amount oflipase was 10 g. The results are shown in table 22. TABLE 22 Resultsobtained with reduced lipase dosage after transesterification usingvacuum and water addition Reaction time PC/LPC/GPC* EPA/DHA** Acid value1 day 90/10/0 0.9/0.7 29 2 days 74/24/2 2.1/1.5 87 3 days 49/27/244.6/4.4 102 6 days 25/32/43 6.7/6.9 115*ELSD peak area (total 100%). Only peaks relating to PC, LPC and GPC areintegrated.**EPA/DHA attached to PC + LPC

EXAMPLE 10

The enzymes from example 7 were isolated by filtration and thepossibility of reuse was determined in the following experiment. 30 gFAEE (10-50), 10 g Alcolec and 15 g used enzymes (equivalent to 7.5 genzyme because the used enzymes had absorbed product from the firstreaction). The reaction was performed using the same conditions as inexample 3. See table 23 below for results. TABLE 23 Reuse of enzymesfrom example 7 using vapor addition into evacuated reaction vessel.Reaction time PC/LPC/GPC* EPA/DHA** Acid value 1 day 79/17/4 1.0/0.9 552 days 59/31/10 2.8/2.6 76 3 days 52/34/14 3.8/3.5 85 6 days 37/43/205.6/5.7 95*ELSD peak area (total 100%). Only peaks relating to PC, LPC and GPC areintegrated.**EPA/DHA attached the fraction consisting of PC + LPC

EXAMPLE 11

The same conditions as in example 7 are applied, except that thepressure in the reaction vessel is 1 mbar. The results obtained aresimilar to the results in Table 23, except that the hydrolysis and theacid values are reduced. After 6 days the relationship between PCspecies is 80/10/0 and the acids value is 40. The incorporation ofEPA/DHA is the same.

EXAMPLE 12

The safety of omega-3 rich phospholipids prepared in the presence ofchloroform and omega-7 rich phospholipids prepared under solvent freeconditions is to be examined by feeding pregnant rats for 1 week. It isto be found that the treatment containing omega-3 rich phospholipds withtraces of chloroform will result in damage to the developing fetus thanthe treatment containing essentially no traces of organic solvents.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

REFERENCES

[1] WO 2006054183

[2] P. C. Calder. Prostaglandins, Leukotrienes and Essential Fatty Acids2006; 75; 197-202.

[3] U.S. Pat. No. 5,434,183

[4] Alexander J W. Nutrition 14 (1998) 627.

[5] Belluzi A, Boschi S, Brignola C, Munarini A, Cariani G and Miglio F.Am J Clin Nutr 71 (2000) 339.

[6] Kremer J M. Am J Clin Nutr 71 (2000) 249

[7] V P Carnielli, G. Verlato, F. Pederzini, I. Luijendijk, A. Boerlage,D. Pedrotti and P. Sauer Am J Clin Nutr 1998;67; 97-103.

[8] M. Moya, E. Cortes, M. Juste, J. G. De Dios and A Vera Eur. J. Clin.Nutr. 2001; 55; 755-762.

[9] A. Sala-Vila, A. I. Castello, C. Campoy, M. Rivero, M.Rodriguez-Palermoe and M. C. Lopez-Sabater. J. Nutr. 2004; 134; 868-873.

[10] A. Sala-Vila, C. Campoy, A. I. Castellote, F. J. Garrido, M.Rivero, M. Rodriguez-Palmero and M. C. Lopez-Sabater. Prostaglandins,Leukotrienes and Essential Fatty Acids; 2006; 74; 143-148.

[11] A. Valenzuela, S. Nieto, J. Sanhueza, M. J. Nunez and C. Ferrer.Annals of Nutrition & Metabolism; 2005; 49; 325-332.

[12] J. B. Hansen, S: Grimsgaard, H. Nilsen, A. Nordoy and K. H. Bonaa.Lipids; 1998; 33; 131-138.

[13] U.S. provisional application entitled “Functional PhospholipidCompositions” with Ser. No. 60/798,027 filed May 5, 2006.

[14] P. C Calder, Philip C. Am. J. Clin. Nutr; 2006; 83; 1505S-1519S.

[15] G. G. Haraldsson, A. Thorarensen, JAOCS 75 (1999) 1143-1149.

[16] G. Lepage and C. C. Roy; J. Lipid Res; 1986; 27; 114-120.

[17] T. Moriguchi, S-Y Lim, R. Greiner, W. Lefkowitz, J. Loewke, J.Hoshiba and N. Salem. J. Lipid Res; 2004;. 45; 1437-1445.

[18] H. Salman, M. Bergman, H Bessler, S Alexandrova, B. Beilin, M.Djaldetti. Acta Phys Scand; 2000; 168, 431-436.

[19] Haraldsson G G and Thorarensen A, JAOCS 75 (1999) 1143.

[20] Samey D B, Fregapane G and Vulfson E N. JAOCS 71 (1994) 93.

1. A composition comprising phospholipids having the followingstructure:

wherein R1 is OH or a fatty acid, R2 is OH or a fatty acid, and R3 is amixture of H, choline, ethanolamine, inositol and serine, saidphospholipid having at least 1% of DHA/EPA at positions R1 and/or R2 andfrom about 20-50% of OH at positions R1 and/or R2.
 2. The composition asclaimed in claim 1, wherein said composition is acylated in a range fromabout 55% to about 85%.
 3. The composition as claimed in claim 2,wherein said composition has a ratio of EPA/DHA ranging from 1:1 to 4:1.4. The composition of claim 2, wherein said composition having a ratioof EPA/DHA ranging from 2:1 to 4:1.
 5. The composition of claim 1,wherein said composition further comprises a lipid carrier in a ratio offrom 1:10 to 10:1 to said phospholipids.
 6. The composition of claim 5,wherein said lipid carrier is selected from the group consisting of atriglyceride, a diglyceride, an ethyl ester, and a methyl ester andcombinations thereof.
 7. The composition in claim 1, wherein saidcomposition provides higher uptake of omega-3 fatty acids into plasma ascompared to natural marine phospholipids.
 8. The composition in claim 1,wherein said composition improves the AA/EPA ratio in plasmaphospholipids as compared to natural marine phospholipids.
 9. Thecomposition in claim 1, wherein said composition increases theconcentration of omega-3 fatty acids in tissues as compared to naturalmarine phospholipids.
 10. The composition in claim 1, wherein saidcomposition reduces the concentration of biomarkers of inflammation ascompared to natural marine phospholipids.
 11. A food product comprisingthe composition in claim
 1. 12. An animal feed comprising thecomposition in claim
 1. 13. A food supplement comprising the compositionin claim
 1. 14. A pharmaceutical comprising the composition in claim 1.15. The composition of claim 5, wherein said lipid carrier and saidphospholipids are in a ratio of from about 5:1 to 1:5.
 16. Thecomposition of claim 5, wherein said composition comprises from about20% to about 90% of said phospholipid composition and from about 10% toabout 50% of said lipid carrier.
 17. A method of preparing abioavailable omega-3 fatty acid composition comprising: a) providing apurified phospholipid composition comprising omega-3 fatty acid residuesand a purified triglyceride composition comprising omega-3 fatty acidresidues; b) combining said phospholipid composition and saidtriglyceride composition to form a bioavailable omega-3 fatty acidcomposition.
 18. The method of claim 17, further comprising the step ofencapsulating said bioavailable omega-3 fatty acid composition.
 19. Themethod of claim 17, wherein said bioavailable omega-3 fatty acidcomposition has increased bioavailability as compared to purifiedtriglycerides or phospholipids comprising omega-3 fatty acid residues.20. The method of claim 17, further comprising the step of packaging thebioavailable omega-3 fatty acid composition for use in functional foods.21. The method of claim 17, further comprising the step of assaying thebioavailable omega-3 fatty acid composition for bioavailability.
 22. Themethod of claim 17, further comprising administering the bioavailableomega-3 fatty acid composition to a patient.
 23. A food productcomprising a bioavailable omega-3 fatty acid composition made theprocess of claim
 17. 24. An animal feed comprising a bioavailableomega-3 fatty acid composition made the process of claim
 17. 25. A foodsupplement comprising a bioavailable omega-3 fatty acid composition madethe process of claim
 17. 26. A pharmaceutical comprising a bioavailableomega-3 fatty acid composition made the process of claim 17.